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Sample records for stably transfected mammalian

  1. Persistent human cardiac Na+ currents in stably transfected mammalian cells

    Science.gov (United States)

    Wang, Ging Kuo; Russell, Gabriella; Wang, Sho-Ya

    2013-01-01

    Miniature persistent late Na+ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na+ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na+ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na+ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na+ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na+ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na+ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na+ currents were blocked by tetrodotoxin with an IC50 value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na+ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na+ currents are suitable for studying as well as screening potent open-channel blockers. PMID:23695971

  2. Persistent human cardiac Na+ currents in stably transfected mammalian cells: Robust expression and distinct open-channel selectivity among Class 1 antiarrhythmics.

    Science.gov (United States)

    Wang, Ging Kuo; Russell, Gabriella; Wang, Sho-Ya

    2013-01-01

    Miniature persistent late Na(+) currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na(+) currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na(+) channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na(+) currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na(+) currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na(+) currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na(+) channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na(+) currents were blocked by tetrodotoxin with an IC(50) value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na(+) currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na(+) currents are suitable for studying as well as screening potent open-channel blockers.

  3. Characterization of cell lines stably transfected with rubella virus replicons

    International Nuclear Information System (INIS)

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-01-01

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  4. Characterization of cell lines stably transfected with rubella virus replicons

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    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  5. Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

    Directory of Open Access Journals (Sweden)

    Wagner G dos Santos

    2000-01-01

    Full Text Available Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

  6. Establishment of Stably Transfected Cells Constitutively Expressing the Full-Length and Truncated Antigenic Proteins of Two Genetically Distinct Mink Astroviruses

    DEFF Research Database (Denmark)

    Bidokhti, Mehdi R. M.; Ullman, Karin; Jensen, Trine Hammer

    2013-01-01

    to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals...

  7. Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Li, Hsiu-Chuan; Huang, Chuan-Chuan; Chen, Sung-Fang; Chou, Min-Yuan

    2005-10-21

    We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.

  8. Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

    Science.gov (United States)

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...

  9. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    Science.gov (United States)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  10. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    Science.gov (United States)

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-12-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10-55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

  11. Photo-transfection of mammalian cells via femtosecond laser pulses

    CSIR Research Space (South Africa)

    Mthunzi, P

    2009-06-01

    Full Text Available ). Transfection efficiencies between 40 - 63 % are recorded. We show for the first time that, due to their different sensitivity, surface receptors and membrane structure the cell lines mentioned above displayed varying photo-transfection efficiencies at different...

  12. THE EVALUATION OF PEPTIDE/HISTIDINE TRANSPORTER 1 (PHT1) FUNCTION: UPTAKE KINETICS UTILIZING A COS-7 STABLY TRANSFECTED CELL LINE.

    Science.gov (United States)

    Lindley, David J; Carl, Stephen M; Mowery, Stephanie A; Knipp, Gregory T

    2011-10-01

    There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H + -dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

  13. GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

    International Nuclear Information System (INIS)

    Morgan, Kevin; Meyer, Colette; Miller, Nicola; Sims, Andrew H; Cagnan, Ilgin; Faratian, Dana; Harrison, David J; Millar, Robert P; Langdon, Simon P

    2011-01-01

    Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R) activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed. GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125 I-ligand binding and stimulation of 3 H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting. GnRH-R immunoscoring was highest in hormone receptor (triple) negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231). After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3 H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation. Breast cancers exhibit a range of GnRH-R immunostaining, with higher levels of

  14. Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

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    Schmidhauser, C. Bissell, M.J. (Univ. of California, Berkeley (United States)); Myers, C.A.; Casperson, G.F. (Monsanto Corporate Research, St. Louis, MO (United States))

    1990-12-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

  15. Optical sorting and photo-transfection of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-02-01

    Full Text Available ), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly...

  16. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    Science.gov (United States)

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  17. SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa Stably Transfected with SPARC.

    Directory of Open Access Journals (Sweden)

    Andrea Slusser-Nore

    Full Text Available This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3 and cadmium (Cd(+2-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2-and As(+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3-and Cd(+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF. Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.It was shown that the As(+3-and Cd(+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3-and Cd(+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.Tumor initiating cells isolated from SPARC-transfected As(+3-and Cd(+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

  18. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    International Nuclear Information System (INIS)

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

    2014-01-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

  19. Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

    Science.gov (United States)

    Suphatrakul, Amporn; Yasanga, Thippawan; Keelapang, Poonsook; Sriburi, Rungtawan; Roytrakul, Thaneeya; Pulmanausahakul, Rojjanaporn; Utaipat, Utaiwan; Kawilapan, Yanee; Puttikhunt, Chunya; Kasinrerk, Watchara; Yoksan, Sutee; Auewarakul, Prasert; Malasit, Prida; Charoensri, Nicha; Sittisombut, Nopporn

    2015-10-13

    Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.

    Science.gov (United States)

    Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

    2017-01-01

    Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.

  1. Polyethyleneimine-poly(ethylene glycol)-star-copolymers as efficient and biodegradable vectors for mammalian cell transfection.

    Science.gov (United States)

    Ladewig, Katharina; Xu, Zhi Ping; Gray, Peter; Max Lu, G Q

    2014-07-01

    High molecular weight (MW) polyethyleneimine (PEI) has been successfully used for the transfection of a broad variety of cell lines. In contrast to low MW PEI, which exhibits low transfection efficiencies but also low cytotoxicity, high MW PEI-mediated transfection achieves much higher efficiencies but at the cost of cell viability; therefore its use in commercial scale transfection and clinical application is limited. In this work we address this problem by constructing biodegradable high MW PEI mimics built from low MW PEI building blocks. The end-groups of small 5-arm star polyethylene glycol (PEG) prepolymers were decorated with linear oligo-ethyleneimine (OEI)/PEI arms of various MW via azomethine linkages. The resultant PEI-PEG-star-copolymers were investigated for their ability to complex plasmid DNA. Polymer/DNA complexes were characterized using techniques such as dynamic light scattering and transmission electron microscopy. Having established their cytotoxicity limits, they were tested as gene delivery vehicles for the transfection of suspension adapted Chinese hamster ovary (CHO-S) cells under serum-free conditions and adherent human embryonic kidney cells (HEK293T) in serum containing medium. Our PEI-PEG-star-copolymers showed a reduced cytotoxicity compared to high MW PEI while maintaining the ability to complex plasmid DNA and transfect mammalian cells, with significant transfection efficiencies. The effects of the optimum parameters on the transfection of mammalian cells using such novel polymers are discussed. © 2013 Wiley Periodicals, Inc.

  2. Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

    Directory of Open Access Journals (Sweden)

    Weeks Sara K

    2009-01-01

    Full Text Available Abstract Background The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars. Results A plasmid transfection approach was used to examine the function of the product of CT223 and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that CT223, and, to a lesser extent, adjacent inc genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells. Conclusion These studies suggest that certain Inc proteins block cytokinesis in C. trachomatis-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.

  3. Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

    Science.gov (United States)

    Muroski, Megan E; Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

    2015-01-27

    Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

  4. Effects of recombinant plasmid pEgr-p53 transfected stably in combination with X-irradiation on cell cycle progression and proliferation in human SKOV-3 tumor cells in vitro

    International Nuclear Information System (INIS)

    Dong Lihua; Liu Feng; Li Yanbo; Fu Shibo; Gong Shouliang

    2008-01-01

    Objective: To investigate the effect of recombinant plasmid pEgr-hp53 transfected stably in combination with X-ray irradiation on the cell cycle progression and the proliferation in human SKOV-3 tumor cells. Methods: pEgr-hp53 and pcDNA3.1 packaged with liposome were stably transfected into SKOV-3 cells in vitro. SKOV-3-hp53 and SKOV-3-vect were irradiated with 0, 0.5, 2.0 and 5.0 Gy X-rays, respectively, i.e. 8 experimental groups. The SKOV-3 cell proliferation and the cell cycle progression were measured with flow cytometry and cell growth curve, respectively. Results: Compared with 0 Gy group, the cell counts in SKOV-3- hp53 plus different doses of irradiation groups 2 d after irradiation decreased significantly (P 0 /G 1 cells increased significantly (P 2 /M cells decreased in varying degrees. The cell counts in SKOV-3-hp53 plus irradiation group were significantly lower than those in corresponding SKOV-3-vect plus irradiation group, the cell counts 4-8 d after irradiation with 0.5 Gy, 2 d after 2.0 Gy irradiation and 6 d after 5.0 Gy irradiation decreased significantly (P 0 /G 1 cells increased significantly (P 2 /M cells decreased significantly (P 1 arrest in SKOV-3 cells and inhibits the cell proliferation. Ionizing radiation can activate early growth response-1 (Egr-1) gene promoter and increase the expression of p53 gene, and enhance the inhibition of tumor cell growth. (authors)

  5. Development of an electro-responsive platform for the controlled transfection of mammalian cells

    Science.gov (United States)

    Hook, Andrew L.; Thissen, Helmut W.; Hayes, Jason P.; Voelcker, Nicolas H.

    2005-02-01

    The recent development of living microarrays as novel tools for the analysis of gene expression in an in-situ environment promises to unravel gene function within living organisms. In order to significantly enhance microarray performance, we are working towards electro-responsive DNA transfection chips. This study focuses on the control of DNA adsorption and desorption by appropriate surface modification of highly doped p++ silicon. Silicon was modified by plasma polymerisation of allylamine (ALAPP), a non-toxic surface that sustains cell growth. Subsequent high surface density grafting of poly(ethylene oxide) formed a layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced micron resolution patterns of re-exposed plasma polymer whilst the rest of the surface remained non-fouling. We observed electro-stimulated preferential adsorption of DNA to the ALAPP surface and subsequent desorption by the application of a negative bias. Cell culture experiments with HEK 293 cells demonstrated efficient and controlled transfection of cells using the expression of green fluorescent protein as a reporter. Thus, these chemically patterned surfaces are promising platforms for use as living microarrays.

  6. Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

    OpenAIRE

    Payne, Andrew J; Gerdes, Bryan C; Kaja, Simon; Koulen, Peter

    2013-01-01

    Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expresse...

  7. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  8. Biphasic action of cyclic adenosine 3',5'- monophosphate in gonadotropin-releasing hormone (GnRH) analog-stimulated hormone release from GH3 cells stably transfected with GnRH receptor complementary deoxyribonucleic acid.

    Science.gov (United States)

    Stanislaus, D; Arora, V; Awara, W M; Conn, P M

    1996-03-01

    GH3 cells are a PRL-secreting adenoma cell line derived from pituitary lactotropes. These cells have been stably transfected with rat GnRH receptor complementary DNA to produce four cell lines: GGH(3)1', GGH(3)2', GGH(3)6', and GGH(3)12'. In response to either GnRH or Buserelin (a metabolically stable GnRH agonist), these cell lines synthesize PRL in a cAMP-dependent manner. Only GGH(3)6' cells desensitize in response to persistent treatment with 10(-7) g/ml Buserelin. GGH(3)1', GGH(3)2', and GGH(3)12' cells, however, can be made refractory to Buserelin stimulation by raising cAMP levels either by the addition of (Bu)2cAMP to the medium or by treatment with cholera toxin. In GGH(3) cells, low levels of cAMP fulfill the requirements for a second messenger, whereas higher levels appear to mediate the development of desensitization. The observation that in GGH(3)6' cells, cAMP production persists after the onset of desensitization is consistent with the view that the mechanism responsible for desensitization is distal to the production of cAMP. Moreover, the absence of any significant difference in the amount of cAMP produced per cell in GGH(3)2', GGH(3)6', or GGH(3)12' cells suggests that elevated cAMP production per cell does not explain the development of desensitization in GGH(3)6' cells. We suggest that Buserelin-stimulated PRL synthesis in GGH(3)6' cells is mediated by a different cAMP-dependent protein kinase pool(s) than that in nondesensitizing GGH(3) cells. Such a protein kinase A pool(s) may be more susceptible to degradation via cAMP-mediated mechanisms than the protein kinase pools mediating the Buserelin response in nondesensitizing GGH(3) cells. A similar mechanism has been reported in other systems.

  9. Transfection of siRNAs can alter miRNA levels and trigger non-specific protein degradation in mammalian cells.

    Science.gov (United States)

    Liang, Xue-Hai; Hart, Christopher E; Crooke, Stanley T

    2013-05-01

    Sequence-non-specific effects of siRNAs that alter the expression of non-targeted genes have been reported, including competition of siRNAs with endogenous RISC components. However, the detailed mechanisms and subsequent effects of such competition are not well documented. Here we analyze the competition of miRNAs in mammalian cells with low concentrations of siRNAs, and found that: 1) transfection of different siRNAs in the low nanomolar range used to deplete target RNAs can reduce the levels of miRNAs in different cell types, 2) siRNA transfection results in rapid reduction of Ago2-associated miRNAs concurrent with accumulation of Ago2-bound siRNAs and a significant change in the expression levels of many miRNAs, 3) competition largely depends on Ago2 and not Dicer, 4) microarray analysis showed that the majority of highly expressed miRNAs are reduced, in a siRNA concentration dependent manner, and low abundant miRNAs may be unchanged or repressed and a few miRNAs appear to have increased levels, and 5) consistent with previous studies, the expression levels of mRNAs that are targeted by highly repressed miRNAs are preferentially increased. As a consequence of such competition, we observed that α-tubulin, a substrate of two up-regulated proteases, granzyme B and granzyme M, was rapidly degraded at the protein level upon siRNA transfection. Our results support a model in which transfection of siRNAs can change the levels of many miRNAs by competition for Ago2, leading to altered expression of many miRNA target genes, which can in turn affect downstream gene expression even at the protein level. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

    Science.gov (United States)

    Pahrudin Arrozi, Aslina; Shukri, Siti Nur Syazwani; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Ahmad Damanhuri, Mohd Hanafi; Makpol, Suzana

    2017-11-01

    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

  11. Characterization of new cell line stably expressing CHI3L1 oncogene

    Directory of Open Access Journals (Sweden)

    Chekhonin V. P.

    2011-06-01

    Full Text Available Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1. Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development.

  12. DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

    Science.gov (United States)

    Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

    2015-12-25

    Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

  13. Cholesterol Domains Enhance Transfection

    Science.gov (United States)

    Betker, Jamie L.; Kullberg, Max; Gomez, Joe; Anchordoquy, Thomas J.

    2014-01-01

    The formation of cholesterol domains in lipoplexes has been associated with enhanced serum stability and transfection rates both in cell culture and in vivo. This study utilizes the ability of saturated phosphatidylcholines to promote the formation of cholesterol domains at much lower cholesterol contents than have been utilized in previous work. The results show that lipoplexes with identical cholesterol and cationic lipid contents exhibit significantly improved transfection efficiencies when a domain is present, consistent with previous work. In addition, studies assessing transfection rates in the absence of serum demonstrate that the ability of domains to enhance transfection is not dependent on interactions with serum proteins. Consistent with this hypothesis, characterization of the adsorbed proteins composing the corona of these lipoplex formulations did not reveal a correlation between transfection and the adsorption of a specific protein. Finally, we show that the interaction with serum proteins can promote domain formation in some formulations, and thereby result in enhanced transfection only after serum exposure. PMID:23557286

  14. Stable transfection of Acanthamoeba.

    Science.gov (United States)

    Yin, J; Henney, H R

    1997-03-01

    The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate-DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 micrograms/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU-Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.

  15. Stably-stratified wall-bounded turbulence

    Science.gov (United States)

    Hadi Sichani, Pejman; Zonta, Francesco; Obabko, Aleksandr; Soldati, Alfredo

    2017-11-01

    Stably-stratified (bottom-up cooling) turbulent flows are encountered in a number of industrial applications, environmental processes and geophysical flows. Turbulent entrainment and mixing across density interfaces in terrestrial water bodies (oceans, lakes and rivers) and in industrial heat transfer equipments are just some important examples of stably-stratified flows. In this work we use Direct Numerical Simulation to investigate the fundamental physics of stably-stratified channel turbulence under Boussinesq and Non-Oberbeck-Boussinesq (NOB) conditions. Compared to the neutrally-buoyant case, in the stably-stratified case active turbulence survives only in the near-wall region and coexists with internal gravity waves (IGW) moving in the core region of the channel. This induces a general suppression of turbulence levels, momentum and buoyancy fluxes. Our results show also that NOB effects may be important when the flow is subject to large temperature gradients. The most striking feature observed in case of NOB conditions is the generation of a strong flow asymmetry with possible local flow laminarization in the near wall region.

  16. Graphene based gene transfection

    Science.gov (United States)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  17. Large eddy simulation of stably stratified turbulence

    International Nuclear Information System (INIS)

    Shen Zhi; Zhang Zhaoshun; Cui Guixiang; Xu Chunxiao

    2011-01-01

    Stably stratified turbulence is a common phenomenon in atmosphere and ocean. In this paper the large eddy simulation is utilized for investigating homogeneous stably stratified turbulence numerically at Reynolds number Re = uL/v = 10 2 ∼10 3 and Froude number Fr = u/NL = 10 −2 ∼10 0 in which u is root mean square of velocity fluctuations, L is integral scale and N is Brunt-Vaïsälä frequency. Three sets of computation cases are designed with different initial conditions, namely isotropic turbulence, Taylor Green vortex and internal waves, to investigate the statistical properties from different origins. The computed horizontal and vertical energy spectra are consistent with observation in atmosphere and ocean when the composite parameter ReFr 2 is greater than O(1). It has also been found in this paper that the stratification turbulence can be developed under different initial velocity conditions and the internal wave energy is dominated in the developed stably stratified turbulence.

  18. Infectious alphavirus production from a simple plasmid transfection+

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2011-07-01

    Full Text Available Abstract We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses.

  19. Toward Contactless Biology: Acoustophoretic DNA Transfection

    Science.gov (United States)

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-02-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

  20. Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

    Science.gov (United States)

    Converse, Andrea D; Belur, Lalitha R; Gori, Jennifer L; Liu, Geyi; Amaya, Felipe; Aguilar-Cordova, Estuardo; Hackett, Perry B; McIvor, R Scott

    2004-12-01

    Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into

  1. Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems

    DEFF Research Database (Denmark)

    Selmeczi, Dávid; Hansen, Thomas Steen; Met, Özcan

    2016-01-01

    Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process...

  2. Transfection of Platyhelminthes

    Directory of Open Access Journals (Sweden)

    Bárbara Moguel

    2015-01-01

    Full Text Available Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  3. Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System

    Directory of Open Access Journals (Sweden)

    Jesse B Owens

    2013-01-01

    Full Text Available Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3 capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT, in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection and long-term integration efficiency (~5-fold enhancement following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3–4 Kb, P < 0.001 in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies.

  4. Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

    Science.gov (United States)

    Kalapila, Aley G.; Pegg, Anthony E.

    2009-01-01

    The primary function of O6-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2–3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (≥3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells. PMID:19941875

  5. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    Science.gov (United States)

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  6. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    Science.gov (United States)

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  7. Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes

    DEFF Research Database (Denmark)

    Ye, Q; Wei, Y; Fischer, R

    1997-01-01

    Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for prote...

  8. Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes

    DEFF Research Database (Denmark)

    Ye, Q; Wei, Y; Fischer, R

    1997-01-01

    Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein...

  9. A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye.

    Science.gov (United States)

    Peng, Lin; Xiong, Wendian; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

    2017-05-04

    Enhanced transfection efficiency of transient gene expression (TGE) and electroporation is a useful approach for improvement of recombinant therapeutic proteins in mammalian cells. A novel method is described here in which CHO cells expressing recombinant FVII (rFVII) were labeled with fluorescent dye and analyzed by confocal microscopy. Cells with or without rFVII encoding gene were detectable by flow cytometry. Thus, we were able to distinguish positive cells (with rFVII encoding gene) and quantify their percentages. We evaluated the effects of varying electroporation conditions (voltage, number of repetitions, plasmid amount, carrier DNA) in order to optimize transfection efficiency. The highest transfection efficiency achieved was ∼86%. The method described here allows rapid evaluation of transfection efficiency without co-expression of reporter genes. In combination with appropriate antibodies, the method can be extended to evaluation of transfection efficiency in cells expressing other recombinant proteins.

  10. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    International Nuclear Information System (INIS)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-01-01

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  11. Photoporation and cell transfection using a violet diode laser

    Science.gov (United States)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  12. Causal boundary for stably causal space-times

    International Nuclear Information System (INIS)

    Racz, I.

    1987-12-01

    The usual boundary constructions for space-times often yield an unsatisfactory boundary set. This problem is reviewed and a new solution is proposed. An explicit identification rule is given on the set of the ideal points of the space-time. This construction leads to a satisfactory boundary point set structure for stably causal space-times. The topological properties of the resulting causal boundary construction are examined. For the stably causal space-times each causal curve has a unique endpoint on the boundary set according to the extended Alexandrov topology. The extension of the space-time through the boundary is discussed. To describe the singularities the defined boundary sets have to be separated into two disjoint sets. (D.Gy.) 8 refs

  13. The radio-sensitivity effect of E1A gene transfected by PEI on colon carcinoma cell in vitro

    International Nuclear Information System (INIS)

    Wu Yinxia; Liu Dongfang; Liu Yongbiao; Xu Dongmei; Yao Side; Sheng Kanglong

    2011-01-01

    As a neotype nonviral vector, (Polyethylenimine, PEI) has been studied in gene transfection experiment. This study was investigated the growth inhibition and radio-sensitizing effect of E1A gene transfected by PEI on human colon carcinoma cell in vitro. The PSV-E1A recombinant plasmid, which was designed for high-level expression of E1A gene in a variety of eukaryotic cell lines, was transfected into SW480 cells by PEI. The transfection was confirmed by RT-PCR and G418 was used to get colon carcinoma cells stably expressed E1A gene. The cell growth curve were investigated to observe the growth inhibition induced by E1A gene. The redistributions of cell cycle were analyzed by flow cytometry. Cells before and after transfection were treated with irradiation, then the changes of radiation-sensitivity were tested by MTT assay after 24 h meanwhile the expression of HER-2 gene in SW480 cells before and after transfection was detected by western-blot. As results, (1) the colon carcinoma cells expressed E1A gene was confirmed by G418. (2) The result of RT-PCR demonstrated that PEI could transfect plasmid psv-E1A and the cells could stably express E1A gene. (3) Flow cytometry revealed that E1A gene transfected into human colon carcinoma cell could induce S stage suppression (p<0.001) and G2/M stage arrest (p<0.001). (4) Compared with the Non-transfected cells, the E1A-transfected cells (SW480-E1A cells) grew slowly observed by MTT assay which was used to get the absorbance of SW480 cell and SW480-E1A cell. (5) The radiation-sensitivity of SW480 cells transfected with E1A gene was up-regulated obviously (p<0.001). (6) The E1A gene obviously down-regulated HER-2 protein expression in colon carcinoma cells. Anyway, PEI can transfect plasmid psv-E1A gene which can significantly inhibit the growth rate of SW480 cell. Moreover, it also obviously enhanced the cell sensitivity to irradiation. (authors)

  14. Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

    Science.gov (United States)

    Hagen, Lars; Sharma, Animesh; Aas, Per Arne; Slupphaug, Geir

    2015-01-01

    Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understanding the molecular pathways affected would add value to the interpretation of experimental observations subsequent to cell transfection. Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene®HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. The results were validated by western analysis as well as quantitative RT-PCR and this demonstrated that an essentially identical response was induced in HeLa by transfection using an empty pUC18 vector, which does not contain a mammalian virus promoter, as well as a liposome-based transfection reagent, Lipofectamine(TM)2000. Notably, no induction of the interferon response was observed in HEK293 cells, suggesting that these cells might be preferable to HeLa to avoid undesired off-target effects in transfection studies encompassing interferon-signaling and antiviral responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

    Science.gov (United States)

    Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

    2008-10-01

    In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

  16. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    DEFF Research Database (Denmark)

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork

    2008-01-01

    Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant...... a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...

  17. Stably Expressed Genes Involved in Basic Cellular Functions.

    Directory of Open Access Journals (Sweden)

    Kejian Wang

    Full Text Available Stably Expressed Genes (SEGs whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age in both sexes of F344 rats (n = 4/group; 320 samples. Expression changes (calculated as the maximum expression / minimum expression for each gene of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination, RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics or exogenous agents (e.g., drugs, environmental factors may cause serious adverse effects.

  18. Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

    Science.gov (United States)

    Horibe, Tomohisa; Torisawa, Aya; Akiyoshi, Ryutaro; Hatta-Ohashi, Yoko; Suzuki, Hirobumi; Kawakami, Koji

    2014-02-01

    The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Enhanced efflux of [3H]vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene

    International Nuclear Information System (INIS)

    Hammond, J.R.; Johnstone, R.M.; Gros, P.

    1989-01-01

    Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain [ 3 H]vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in [ 3 H]vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in [ 3 H]vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of [ 3 H]vinblastine release. The observation that the mdr1 transfectants show a decreased [ 3 H]vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for [ 3 H]vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants

  20. Optimal energy growth in a stably stratified shear flow

    Science.gov (United States)

    Jose, Sharath; Roy, Anubhab; Bale, Rahul; Iyer, Krithika; Govindarajan, Rama

    2018-02-01

    Transient growth of perturbations by a linear non-modal evolution is studied here in a stably stratified bounded Couette flow. The density stratification is linear. Classical inviscid stability theory states that a parallel shear flow is stable to exponentially growing disturbances if the Richardson number (Ri) is greater than 1/4 everywhere in the flow. Experiments and numerical simulations at higher Ri show however that algebraically growing disturbances can lead to transient amplification. The complexity of a stably stratified shear flow stems from its ability to combine this transient amplification with propagating internal gravity waves (IGWs). The optimal perturbations associated with maximum energy amplification are numerically obtained at intermediate Reynolds numbers. It is shown that in this wall-bounded flow, the three-dimensional optimal perturbations are oblique, unlike in unstratified flow. A partitioning of energy into kinetic and potential helps in understanding the exchange of energies and how it modifies the transient growth. We show that the apportionment between potential and kinetic energy depends, in an interesting manner, on the Richardson number, and on time, as the transient growth proceeds from an optimal perturbation. The oft-quoted stabilizing role of stratification is also probed in the non-diffusive limit in the context of disturbance energy amplification.

  1. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    International Nuclear Information System (INIS)

    Matsushima, H.; Bogenmann, E.

    1990-01-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment

  2. Mammalian sleep

    Science.gov (United States)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  3. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

    Science.gov (United States)

    Chernousova, S; Epple, M

    2017-05-01

    The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

  4. Tissue Engineering Using Transfected Growth-Factor Genes

    Science.gov (United States)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  5. Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

    International Nuclear Information System (INIS)

    Jeang, K.T.; Cho, M.S.; Hayward, G.S.

    1984-01-01

    A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors

  6. Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization.

    Science.gov (United States)

    Li, Junwei; Yoon, Soon Joon; Hsieh, Bao-Yu; Tai, Wanyi; O'Donnell, Matthew; Gao, Xiaohu

    2015-12-09

    Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core-shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing.

  7. Optimization of renal transfection using a renal suction-mediated transfection method in mice.

    Science.gov (United States)

    Taniguchi, Yota; Kawakami, Shigeru; Fuchigami, Yuki; Oyama, Natsuko; Yamashita, Fumiyoshi; Konishi, Satoshi; Shimizu, Kazunori; Hashida, Mitsuru

    2016-01-01

    We previously developed a suction-mediated transfection method in mice. The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice. Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined. The renal suction-mediated transfection method at -30 kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA). We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.

  8. Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

    Science.gov (United States)

    Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

    2018-02-20

    Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel

  9. Dynamics of mixed convective-stably-stratified fluids

    Science.gov (United States)

    Couston, L.-A.; Lecoanet, D.; Favier, B.; Le Bars, M.

    2017-09-01

    We study the dynamical regimes of a density-stratified fluid confined between isothermal no-slip top and bottom boundaries (at temperatures Tt and Tb) via direct numerical simulation. The thermal expansion coefficient of the fluid is temperature dependent and chosen such that the fluid density is maximum at the inversion temperature Tb>Ti>Tt . Thus, the lower layer of the fluid is convectively unstable while the upper layer is stably stratified. We show that the characteristics of the convection change significantly depending on the degree of stratification of the stable layer. For strong stable stratification, the convection zone coincides with the fraction of the fluid that is convectively unstable (i.e., where T >Ti ), and convective motions consist of rising and sinking plumes of large density anomaly, as is the case in canonical Rayleigh-Bénard convection; internal gravity waves are generated by turbulent fluctuations in the convective layer and propagate in the upper layer. For weak stable stratification, we demonstrate that a large fraction of the stable fluid (i.e., with temperature T phenomenological description of the transition between the regimes of plume-dominated and entrainment-dominated convection through analysis of the differences in the heat transfer mechanisms, kinetic energy density spectra, and probability density functions for different stratification strengths. Importantly, we find that the effect of the stable layer on the convection decreases only weakly with increasing stratification strength, meaning that the dynamics of the stable layer and convection should be studied self-consistently in a wide range of applications.

  10. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    Science.gov (United States)

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

  11. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering...

  12. Optimized PEI-based Transfection Method for Transient Transfection and Lentiviral Production.

    Science.gov (United States)

    Yang, Shaozhe; Zhou, Xiaoling; Li, Rongxiang; Fu, Xiuhong; Sun, Pingnan

    2017-09-14

    Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  13. [RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO].

    Science.gov (United States)

    Zhang, Guangde; Su, Chengshuai; Jin, Xia; Yang, Shimao; Fang, Dianji; Guo, Yanwei

    2016-03-01

    To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the

  14. An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.

    Science.gov (United States)

    Hubner, Eric K; Lechler, Christian; Kohnke-Ertel, Birgit; Zmoos, Anne-Flore; Sage, Julien; Schmid, Roland M; Ehmer, Ursula

    2017-01-01

    Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice. After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system. Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Stable transfection of provirus of human immunodeficiency virus into a murine packaging cell line.

    Science.gov (United States)

    Rozera, C; Baccarini, S; Gentile, M; Torrisi, M R; Proietti, E; Federico, M; Pulciani, S

    1997-04-01

    In order to generate HIV (murine leukemia virus (MuLV)) pseudotypes, HIV genome was transfected into the ecotropic murine packaging cell line (GP+E86) and four of the nine transfected clones were extensively characterized. One clone (801), harbouring a full copy of integrated HIV sequences, exhibited a detectable level of intracellular HIV p24 antigen expression. Northern blot analysis revealed that clone 801 expressed all three classes of HIV mRNAs. Multispliced 2 kb mRNAs were detected in another clone (8.14). Two other clones (1.31 and 1.32) also exhibited a complete HIV provirus, but did not show any viral expression, as evaluated by Northern blot analysis or HIV p24 ELISA. Reverse transcription-polymerase chain reaction (RT-PCR) experiments revealed the presence of full length genomic RNA in four transfected clones, which were extensively characterized. A co-cultivation of clone 801 with human CD4' cells resulted in syncytia formation. By electron microscopy, mature HIV particles were observed after co-cultivation of uninfected C8166 cells with 801 cells. These results demonstrated that the murine clone was stably transfected with the complete HIV genome and was capable of shuttling infectious HIV to human cells. Clone 801 was co-cultivated with murine NIH-3T3 fibroblasts. In several experiments, HIV infection of NIH-3T3 cells was revealed by PCR technique. Thus, 801 cells appear to produce low levels of HIV (MuLV) pseudotypes capable of transferring the HIV genome into mouse cells.

  16. Fibronectin enhances transfection of Staphylococcus aureus.

    OpenAIRE

    Thompson, N E; Bergdoll, M S; Pattee, P A

    1985-01-01

    The factor in normal sera primarily responsible for the enhancement of transfection (and transformation) of Staphylococcus aureus was identified as fibronectin. Serum samples which were depleted of fibronectin by affinity chromatography showed a marked decrease in enhancing activity. Fibronectin isolated from sera of several animal species demonstrated enhancing activity.

  17. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    Science.gov (United States)

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

  18. Suppression of mRNA Nanoparticle Transfection in Human Fibroblasts by Selected Interferon Inhibiting Small Molecule Compounds.

    Science.gov (United States)

    Liu, Yang; Krishnan, Manoj N; Phua, Kyle K L

    2017-07-31

    In vitro transcribed (IVT) mRNA is increasingly applied in lieu of DNA to deliver reprogramming genes to fibroblasts for stem cell derivation. However, IVT mRNA induces interferon (IFN) responses from mammalian cells that reduces transfection efficiency. It has been previously suggested that small molecule inhibitors of IFN are a viable strategy to enhance mRNA transfection efficiency. Herein, we screen a list of commercially available small molecules, including published IFN inhibitors, for their potential to enhance mRNA transfection in BJ fibroblasts. Transfection enhancement is quantified by relative mean fluorescence intensity of translated green fluorescent protein (GFP) in treated cells compared to dimethyl sulfoxide treated controls. Within toxicological constrains, all tested small molecules did not enhance mRNA transfection in BJ fibroblasts while a third of the tested compounds unexpectedly inhibited GFP expression even though IFN-β production is inhibited. Based on the results of our study, we conclude that small molecule inhibitors, including IFN inhibitors, tested in this study do not enhance in vitro mRNA transfection efficiency in human fibroblasts.

  19. Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains.

    Science.gov (United States)

    Wu, Shi-Hao; Liao, Zhi-Xing; D Rizak, Joshua; Zheng, Na; Zhang, Lin-Heng; Tang, Hen; He, Xiao-Bin; Wu, Yang; He, Xia-Ping; Yang, Mei-Feng; Li, Zheng-Hui; Qin, Dong-Dong; Hu, Xin-Tian

    2017-03-18

    Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca 2+ /calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.

  20. Optomizing Transfection Efficiency of Cervical Cancer Cells Transfected by Cationic Liposomes LipofectamineTM2000.

    Science.gov (United States)

    Huang, Fei; Zhao, Feng; Liang, Li-Ping; Zhou, Mei; Qu, Zhi-Ling; Cao, Yan-Zhen; Lin, Chen

    2015-01-01

    Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Lipofectamine(TM)2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. The seeding density of Hela cell line and Siha are 1.5 x 10(4) (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (Ptransfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

  1. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    Directory of Open Access Journals (Sweden)

    Helena Sork

    2016-01-01

    Full Text Available The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  2. A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

    Directory of Open Access Journals (Sweden)

    Zhang Tingting

    2012-12-01

    Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

  3. A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Hanxin Lu

    2011-02-01

    Full Text Available Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330 and HA0(1-500 proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1 as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.

  4. NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

    International Nuclear Information System (INIS)

    Lacoste, J.; Cohen, L.; Hiscott, J.

    1991-01-01

    The effect of constitutive Tax expression on the interaction of NF-κ B with its recognition sequence and on NF-κ B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-κ B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-κ B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters

  5. Mammalian airborne allergens

    NARCIS (Netherlands)

    Aalberse, Rob C.

    2014-01-01

    Historically, horse dandruff was a favorite allergen source material. Today, however, allergic symptoms due to airborne mammalian allergens are mostly a result of indoor exposure, be it at home, at work or even at school. The relevance of mammalian allergens in relation to the allergenic activity of

  6. Optimizing conditions for calcium phosphate mediated transient transfection

    Directory of Open Access Journals (Sweden)

    Ling Guo

    2017-03-01

    Conclusions: Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

  7. The Ig heavy chain switch region is a hotspot for insertion of transfected DNA

    Energy Technology Data Exchange (ETDEWEB)

    Baar, J.; Shulman, M.J. [Univ. of Toronto, Ontario (Canada)

    1995-08-15

    The Ig heavy chain switch usually occurs by breaking and rejoining DNA in the switch (S) regions, which consist of tandemly repeated sequences 5{prime} of the constant region exons. Various studies have suggested that S DNA can also recombine with non-S sequences. To measure the frequency of such recombination events, the hybridoma cell line igm692, a deletion mutant that lacks the C{mu}1 and C{mu}2 exons and the 3{prime} end of the S{mu} region, was transfected with a fragment bearing the C{mu}1-2 exons, but no S{mu} DNA. Insertion of this fragment into the residual VDJ-C{mu} intron of igm692 can restore a functional {mu} gene, yielding a transformant that is detected as a plaque-forming cell (PFC). PFCs comprise {approximately}8 x 10{sup -7} of the surviving transfected cells. In 10 of 12 PFCs, the C{mu}1-2 fragment inserted into the 2.5-kb residual S{mu} region, whereas insertion in two cases occurred in the 3.5-kb segment 5{prime} of S{mu}. Using a PCR assay to measure the frequency of insertion of the tranferred fragment elsewhere in the hybridoma genome, we found that {approximately}9% of the surviving tranfected cells had stably acquired the C{mu}1-2 fragment. These results indicate that the S{mu} region is {approximately}100-fold more recombinogenic than the average genomic site, and {approximately}7-fold more recombinogenic than the non-S{mu} segment of the residual VDJ-C{mu}, i.e., the S{mu} region is a hotspot for insertion of transfected DNA.

  8. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    CSIR Research Space (South Africa)

    Mthunzi, P

    2014-02-01

    Full Text Available stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial...

  9. Microfluidic platforms for high-throughput mammalian cell printing, transfection, and dosage-dependent studies

    OpenAIRE

    Woodruff, Kristina Pan

    2017-01-01

    With the advent of high-throughput and genome-wide screening initiatives, there is a need for improved methods for cell-based assays. Current approaches require expensive equipment, rely on large-scale culturing formats not suited for small or rare sample types, or involve tedious manual handling. Microfluidic systems could provide a solution to these limitations, since these assays are accessible, miniaturized, and automated. When coupled with high-content analysis, microfluidics has the pot...

  10. Kvbeta2 inhibits the Kvbeta1-mediated inactivation of K+ channels in transfected mammalian cells.

    Science.gov (United States)

    Xu, J; Li, M

    1997-05-02

    Cloned auxiliary beta-subunits (e.g. Kvbeta1) modulate the kinetic properties of the pore-forming alpha-subunits of a subset of Shaker-like potassium channels. Coexpression of the alpha-subunit and Kvbeta2, however, induces little change in channel properties. Since more than one beta-subunit has been found in individual K+ channel complexes and expression patterns of different beta-subunits overlap in vivo, it is important to test the possible physical and/or functional interaction(s) between different beta-subunits. In this report, we show that both Kvbeta2 and Kvbeta1 recognize the same region on the pore-forming alpha-subunits of the Kv1 Shaker-like potassium channels. In the absence of alpha-subunits the Kvbeta2 polypeptide interacts with additional beta-subunit(s) to form either a homomultimer with Kvbeta2 or a heteromultimer with Kvbeta1. When coexpressing alpha-subunits and Kvbeta1 in the presence of Kvbeta2, we find that Kvbeta2 is capable of inhibiting the Kvbeta1-mediated inactivation. Using deletion analysis, we have localized the minimal interaction region that is sufficient for Kvbeta2 to associate with both alpha-subunits and Kvbeta1. This mapped minimal interaction region is necessary and sufficient for inhibiting the Kvbeta1-mediated inactivation, consistent with the notion that the inhibitory activity of Kvbeta2 results from the coassembly of Kvbeta2 with compatible alpha-subunits and possibly with Kvbeta1. Together, these results provide biochemical evidence that Kvbeta2 may profoundly alter the inactivation activity of another beta-subunit by either differential subunit assembly or by competing for binding sites on alpha-subunits, which indicates that Kvbeta2 is capable of serving as an important determinant in regulating the kinetic properties of K+ currents.

  11. Hydrophobic modification of polyethyleneimine for gene transfectants

    International Nuclear Information System (INIS)

    Kim, Sung Tae; Choi, Joon Sig; Jang, Hyung Suk; Suh, Hea Ran; Park, Jong Sang

    2001-01-01

    A new gene transfer system was developed by using polylipoplexes, which were prepared by hydrophobic modification of polyethyleneimine (PEI, MW 2000). PEI 25kDa is well known for its excellent transfection efficiency but it has extreme cytotoxicity; therefore, its application for medical use is strictly limited. PEI 2kDa is able to form complexes with DNA and has low cytotoxicity. However, unfortunately, it shows no transfection efficiency so it can not be a candidate carrier for gene therapy. We designed novel polycationic amphilphiles by conjugating hydrophobic moieties, such as cholesterol and myristate, to PEI 2kDa. Cholesterol-conjugated PEI (PEI-Chol: P10C, P17C and P30C) and myristate-conjugated PEI (PEI-Myr:P10M, P16M and P26M) are different from the other cationic lipids in that they can form lipopolyplexes with plasmid DNA that have extra multi-positive charges in their hydrophilic parts. From a different point of view, they are also considered to be PEI derivatives with a small proportion of hydrophobic moiety. As a result of the modification, PEI-Chol and PEI-Myr showed much enhanced transfection activity but somewhat increased cytotoxicity. We also examined the effect of the amount of hydrophobic moiety on lipopolyplex-mediated gene transfer and observed that P17C and P26M are the most effective carriers in the series of two groups. MTT assay indicated that the more myristyl groups were attached to PEI, the more injurious results were observed. In the case of PEI-Chol, however, the opposite tendency was observed

  12. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc.

  13. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-02-01

    Full Text Available Abstract Background Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP using PEI. Results Optimal transfection efficiency was observed with 1 μg/ml of plasmid DNA and 5 μg/ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. Conclusions These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

  14. Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

    Science.gov (United States)

    Rheiner, Steven; Reichel, Derek; Rychahou, Piotr; Izumi, Tadahide; Yang, Hsin-Sheng; Bae, Younsoo

    2017-08-07

    Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'transfection in the luciferase assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

    International Nuclear Information System (INIS)

    Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F.

    1988-01-01

    The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression

  16. A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.

    Science.gov (United States)

    Figueroa, Elizabeth; Bugga, Pallavi; Asthana, Vishwaratn; Chen, Allen L; Stephen Yan, J; Evans, Emily Reiser; Drezek, Rebekah A

    2017-05-02

    Gold-polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au-PAMAM transfection than others. Here we utilized two representative cell lines-a "difficult to transfect" CT26 cell line and an "easy to transfect" SK-BR3 cell line-and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.

  17. RNAa is conserved in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Vera Huang

    2010-01-01

    Full Text Available RNA activation (RNAa is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed 'small activating RNAs' (saRNAs. Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals.In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee, mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1 and chimpanzee (WES cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose-derived stem cells.This study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.

  18. A germline chromothripsis event stably segregating in 11 individuals through three generations

    DEFF Research Database (Denmark)

    Bertelsen, Birgitte; Nazaryan-Petersen, Lusine; Sun, Wei

    2016-01-01

    PURPOSE: Parentally transmitted germ-line chromothripsis (G-CTH) has been identified in only a few cases. Most of these rearrangements were stably transmitted, in an unbalanced form, from a healthy mother to her child with congenital abnormalities probably caused by de novo copy-number changes of...

  19. A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

    Science.gov (United States)

    Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

    2015-08-01

    Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

  20. [Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line].

    Science.gov (United States)

    Gan, Zhi-Hua; Chen, Yu; Yan, Hua; Wang, Kan-Kan

    2010-08-01

    Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.

  1. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Science.gov (United States)

    Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI). PMID:28239294

  2. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Konrad H. Engelhardt

    2017-01-01

    Full Text Available Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc at certain nitrogen-to-phosphorus (N/P ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3. Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS, atomic force microscopy (AFM, and scanning electron microscopy (Cryo-SEM, respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI.

  3. Repeated Aurora-A siRNA Transfection Results in Effective Apoptosis of A549 Cells Compared to Single Transfection.

    Science.gov (United States)

    Wang, Zhonghua; Sun, Wenwu; Cao, Jianping; Cui, Haiyang; Ma, Zhuang

    2016-01-01

    Suppression of Aurora kinase A (Aurora-A, AURKA) by Aurora-A siRNA has been proposed for lung tumor treatment. However, protocols using single administration have shown little benefit in some types of lung tumor. Given that transfection efficiency of Aurora-A siRNA is low due to tightly packed cells in the tumor, we hypothesized that repeated administration would result in efficient cell apoptosis. We compared single vs. repeated transfection (thrice) in A549 cells by transfecting Aurora-A siRNA (siA) on the 1st or 1st, 2nd and 3rd day after cell seeding. A random sequence was used as the negative siRNA control (siC). Cells in the single transfection group received only transfection reagent without siRNAs on the 2nd and 3rd day. Two days after the third transfection, both single and repeated siA administration decreased mRNA expression of Aurora-A and cell viability compared to no administration and siC single administration. However, the decrease in these two indices with repeated transfection was more obvious than that following single administration: cell viability decreased to 72.8 ± 3.05% (p transfection and to 64.2 ± 1.99% (p transfection, compared with normal control cells, respectively. Gene expression decreased to 17 ± 16.6% (p transfection and to 43.2 ± 13.0% (p transfection. Compared to single transfection, repeated Aurora-A siRNA transfection decreased Aurora-A, which, in turn, resulted in effective apoptosis of A549 cells.

  4. Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

    Directory of Open Access Journals (Sweden)

    Takuro Horii

    2013-12-01

    Full Text Available Haploid embryonic stem cells (ESCs are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%. Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.

  5. pEGFP transfection into murine skeletal muscle by electrosonoporation

    Science.gov (United States)

    Tamošiūnas, Mindaugas; Jakovels, Dainis; Rubins, Uldis; Kadikis, Roberts; Petrovska, Ramona; Šatkauskas, Saulius

    2017-12-01

    In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can affect the plasmid DNA transfection to mice tibialis cranialis muscle. Multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) fluorescence, providing information on location and duration of EGFP expression. We found that electrosonoporation, commonly enhancing pDNA transfection in vitro, had no positive effect on EGFP transfection efficiency increase in vivo with respect to electroporation alone. We presume that this may be associated with decreased viability of transfected fibers.

  6. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...... transfected. Parvalbumin-transfected and mock-transfected cells were loaded with the calcium indicator fura-2 and were exposed, in the same dish, to different concentrations of the calcium ionophore A23187 or to KCI. The results show that parvalbumin-transfected PCC7 cells had much better calcium buffering...

  7. Inducement of radionuclides targeting therapy by gene transfection

    International Nuclear Information System (INIS)

    Luo Quanyong

    2001-01-01

    The author presents an overview of gene transfection methods to genetically induce tumor cells to express enhanced levels of cell surface antigens and receptors to intake radiolabeled antibody and peptide targeting and thus increase their therapeutic effect in radiotherapy. The current research include inducement of radioimmunotherapy through CEA gene transfection, inducement of iodine-131 therapy by sodium iodide symporter gene transfection and inducement of MIBG therapy by noradrenaline transporter gene transfection. These studies raise the prospect that gene-therapy techniques could be used to enable the treatment of a wide range of tumors with radiopharmaceuticals of established clinical acceptability

  8. Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

    International Nuclear Information System (INIS)

    Hashiguchi, Kohtaro; Ozaki, Masumi; Kuraoka, Isao; Saitoh, Hisato

    2013-01-01

    Highlights: ► A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. ► Nip45 does not effectively inhibit polySUMOylation in vivo. ► Nip45 interacts directly with SUMO and SUMO chains. ► Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

  9. An assessment of the overexpression of BMP-2 in transfected human osteoblast cells stimulated by mineral trioxide aggregate and Biodentine.

    Science.gov (United States)

    Rodrigues, E M; Gomes-Cornélio, A L; Soares-Costa, A; Salles, L P; Velayutham, M; Rossa-Junior, C; Guerreiro-Tanomaru, J M; Tanomaru-Filho, M

    2017-12-01

    To evaluate the effect of MTA and Biodentine on viability, osteogenic differentiation and BMP-2 expression in osteogenic cells. Saos-2 cells were used as a model of osteoblastic cells. Overexpression of BMP-2 was induced by transfection of a CMV-driven plasmid construct including the human BMP-2 coding sequence, and stably transfected cells were selected. Cell viability was assessed by the mitochondrial dehydrogenase enzymatic (MTT) assay. The bioactivity of the materials was evaluated by the alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2 and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). Viability tests revealed that MTA and Biodentine were not cytotoxic at the higher dilution (1 : 8) to BMP-2-transfected cells. MTA and Biodentine exhibited the highest ALP activity when compared to the Saos-BMP-2-unexposed control group (P Biodentine and MTA had a significant stimulatory effect on the formation of mineralized nodules (P Biodentine in non-osteogenic medium in relation to Saos-BMP-2-unexposed control cells (P Biodentine showed biocompatibility and bioactivity in Saos-BMP-2 overexpressing cells. Biodentine had a significantly greater effect on mineralization than MTA. Both MTA and Biodentine enhanced BMP-2 mRNA expression in the transfected system. Both MTA and Biodentine are suitable materials to improve osteoblastic cell mineralization. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  10. Mammalian cell biology

    International Nuclear Information System (INIS)

    Elkind, M.M.

    1975-01-01

    Progress is reported on the following research projects: the effects of N-ethyl-maleimide and hydroxyurea on hamster cells in culture; sensitization of synchronized human cells to x rays by N-ethylmaleimide; sensitization of hypoxic mammalian cells with a sulfhydryl inhibitor; damage interaction due to ionizing and nonionizing radiation in mammalian cells; DNA damage relative to radioinduced cell killing; spurious photolability of DNA labeled with methyl- 14 C-thymidine; radioinduced malignant transformation of cultured mouse cells; a comparison of properties of uv and near uv light relative to cell function and DNA damage; Monte Carlo simulation of DNA damage and repair mechanisms; and radiobiology of fast neutrons

  11. [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].

    Science.gov (United States)

    Li, Ji; Liu, Zuojin

    2015-12-01

    To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (Ptransfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.

  12. Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells.

    Science.gov (United States)

    Uni, Z; Pratt, W D; Miller, M M; O'Connell, P H; Schat, K A

    1994-12-01

    Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes (B19B19 and B13B13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a (B19B19) and S13 (B13B13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

  13. Improved transfection of HUVEC and MEF cells using DNA ...

    Indian Academy of Sciences (India)

    Cells such as mouse embryonic fibroblasts (MEFs) and human umbilical vein endothelial cells (HUVECs) used in stem cell research and endothelial cell physiology and pathology studies are difficult to transfect using 'standard' nonviral transfection methods. We have developed a novel gene delivery technique, which uses ...

  14. Correlation between cationic lipid-based transfection and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael, E-mail: michael@elbaum.ac.il

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

  15. Building the mammalian testis

    DEFF Research Database (Denmark)

    Svingen, Terje; Koopman, Peter

    2013-01-01

    Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial...

  16. Mammalian Antibiotic Peptides

    Czech Academy of Sciences Publication Activity Database

    Šíma, Petr; Trebichavský, Ilja; Sigler, Karel

    2003-01-01

    Roč. 48, č. 2 (2003), s. 123-137 ISSN 0015-5632 R&D Projects: GA ČR GA301/02/1232; GA ČR GA524/01/0917 Institutional research plan: CEZ:AV0Z5020903 Keywords : mammalian Subject RIV: EE - Microbiology, Virology Impact factor: 0.857, year: 2003

  17. Correlation between cationic lipid-based transfection and cell division.

    Science.gov (United States)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. Copyright © 2016. Published by Elsevier Inc.

  18. Highly efficient transfection of human THP-1 macrophages by nucleofection.

    Science.gov (United States)

    Maeß, Marten B; Wittig, Berith; Lorkowski, Stefan

    2014-09-02

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

  19. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    Science.gov (United States)

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    Directory of Open Access Journals (Sweden)

    Roberts Paul C

    2009-04-01

    Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

  1. HTLV-1 Tax mediated downregulation of miRNAs associated with chromatin remodeling factors in T cells with stably integrated viral promoter.

    Directory of Open Access Journals (Sweden)

    Saifur Rahman

    Full Text Available RNA interference (RNAi is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1 transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR using a CD4(+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

  2. Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

    OpenAIRE

    Perkins, J B; Dean, D H

    1983-01-01

    DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.

  3. Acoustic Liquid Handling for Rapid siRNA Transfection Optimization.

    Science.gov (United States)

    Xiao, Andrew S; Lightcap, Eric S; Bouck, David C

    2015-09-01

    Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues. © 2015 Society for Laboratory Automation and Screening.

  4. Enhancement of DNA-transfection frequency by X-rays

    International Nuclear Information System (INIS)

    Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi

    1997-01-01

    This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

  5. Mammalian Synthetic Biology

    OpenAIRE

    Martella, Andrea; Pollard, Steven M; Dai, Junbiao; Cai, Yizhi

    2016-01-01

    The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-pote...

  6. Rheotaxis guides mammalian sperm

    Science.gov (United States)

    Miki, Kiyoshi; Clapham, David E

    2013-01-01

    Background In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals. Results Here, we show that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated within 4 hours of sexual stimulation and coitus in female mice; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement is demonstrated in capacitated and uncapacitated spermatozoa in low and high viscosity medium. Finally, we show that a unique sperm motion we quantify using the sperm head's rolling rate reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels, presumably by enabling Ca2+ influx. Conclusions We propose that rheotaxis is a major determinant of sperm guidance over long distances in the mammalian female reproductive tract. Coitus induces fluid flow to guide sperm in the oviduct. Sperm rheotaxis requires rotational motion during CatSper channel-dependent hyperactivated motility. PMID:23453951

  7. Photobiomodulation on KATP Channels of Kir6.2-Transfected HEK-293 Cells

    Directory of Open Access Journals (Sweden)

    Fu-qing Zhong

    2014-01-01

    Full Text Available Background and Objective. ATP-sensitive potassium (KATP channel couples cell metabolism to excitability. To explore role of KATP channels in cellular photobiomodulation, we designed experiment to study effect of low intensity 808 nm laser irradiation on the activity of membrane KATP channel. Study Design/Materials and Methods. Plasmids encoding Kir6.2 was constructed and heterologously expressed in cultured mammalian HEK-293 cells. The patch-clamp and data acquisition systems were used to record KATP channel current before and after irradiation. A laser beam of Ga-As 808 nm at 5 mW/cm2 was used in experiments. A one-way ANOVA test followed by a post hoc Student-Newman-Keuls test was used to assess the statistical differences between data groups. Results. Obvious openings of KATP channels of Kir6.2-transfected HEK-293 cells and excised patches were recorded during and after low intensity 808 nm laser irradiation. Compared with the channels that did not undergo irradiation, open probability, current amplitude, and dwell time of KATP channels after irradiation improved. Conclusions. Low intensity 808 nm laser irradiation may activate membrane KATP channels of Kir6.2-transfected HEK-293 cells and in excised patches.

  8. Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells.

    Science.gov (United States)

    Palchetti, Sara; Pozzi, Daniela; Marchini, Cristina; Amici, Augusto; Andreani, Cristina; Bartolacci, Caterina; Digiacomo, Luca; Gambini, Valentina; Cardarelli, Francesco; Di Rienzo, Carmine; Peruzzi, Giovanna; Amenitsch, Heinz; Palermo, Rocco; Screpanti, Isabella; Caracciolo, Giulio

    2017-02-01

    To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Reduction of vertical transport in two-dimensional stably stratified forced shear flows

    Science.gov (United States)

    Toqué, Nathalie; Lignières, François; Vincent, Alain

    2006-04-01

    The effect of stable stratification on the vertical transport of passive contaminants in forced, stationary, two-dimensional (2D) and inhomogeneous shear turbulence is investigated numerically. The mean flow consists of several superimposed parallel sheared layers in a stably stratified medium. We find that, as stratification increases, the vertical transport decreases much faster than predicted by mixing length estimates. For the highest stratification, particles vertical dispersion nearly vanishes. The proposed interpretation emphasizes the role of weakly sheared layers where the relative increase of the mean horizontal velocity with respect to the root-mean-square (rms) vertical velocity causes the decrease of the Lagrangian correlation timescale.

  10. Enhanced production of docosahexaenoic acid in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Guiming Zhu

    Full Text Available Docosahexaenoic acid (DHA, one of the important polyunsaturated fatty acids (PUFA with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA. However, the accumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased production of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.

  11. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    Directory of Open Access Journals (Sweden)

    Lin-Lin Liu

    Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  12. Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL

    Science.gov (United States)

    Zustiak, Matthew P.; Jose, Lisa; Xie, Yueqing; Zhu, Jianwei; Betenbaugh, Micheal J.

    2014-01-01

    Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming. PMID:24604826

  13. Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications.

    Science.gov (United States)

    Soto-Sánchez, Cristina; Martínez-Navarrete, Gema; Humphreys, Lawrence; Puras, Gustavo; Zarate, Jon; Pedraz, José Luis; Fernández, Eduardo

    2015-05-01

    This work demonstrates the successful long-term transfection in vivo of a DNA plasmid vector in rat visual cortex neurons using the magnetofection technique. The transfection rates reached values of up to 97% of the neurons after 30days, comparable to those achieved by viral vectors. Immunohistochemical treatment with anti-EGFP antibodies enhanced the detection of the EYFP-channelrhodopsin expression throughout the dendritic trees and cell bodies. These results show that magnetic nanoparticles offer highly efficient and enduring in vivo high-rate transfection in identified neurons of an adult mammalian brain and suggest that the magnetotechnique facilitates the introduction of large functional genetic material like channelrhodopsin with safe non-viral vectors using minimally invasive approaches. Gene therapy may be one of the treatment modalities for neurological diseases in the future. The use of viral transfection remains a concern due to restrictions to the size limit of the genetic material able to be packed, as well as safety issues. In this work, the authors evaluated magnetoplexes as an alternative vehicle. The results showed very promising data in that these nanoparticles could offer high transfection efficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

    International Nuclear Information System (INIS)

    Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong

    2007-01-01

    The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation

  15. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...... that depletion of ACBP in mammalian cells results in lethality, suggesting that ACBP is an essential protein....

  16. Codon-Optimized Luciola Italica Luciferase Variants for Mammalian Gene Expression in Culture and in Vivo

    Directory of Open Access Journals (Sweden)

    Casey A. Maguire

    2012-01-01

    Full Text Available Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-D-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.

  17. A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293.

    Science.gov (United States)

    Jain, Nina K; Barkowski-Clark, Susan; Altman, Richard; Johnson, Krista; Sun, Fang; Zmuda, Jonathan; Liu, Chao Yan; Kita, Adriana; Schulz, Ryan; Neill, Alyssa; Ballinger, Robert; Patel, Rekha; Liu, Jian; Mpanda, Alinafe; Huta, Brian; Chiou, Henry; Voegtli, Walter; Panavas, Tadas

    2017-06-01

    Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins. Therefore, it is potentially advantageous to keep the host cell type consistent throughout drug discovery and development. To this end, we compared the ExpiCHO system, a high density CHO-S transient transfection system, to the Expi293 and FreeStyle MAX CHO transient systems. Fourteen proteins were expressed in both the Expi293 and ExpiCHO systems. For a majority of proteins tested, the protein titers observed with the ExpiCHO system were higher than those seen with both the FreeStyle MAX CHO and Expi293 systems. Antibodies expressed using the ExpiCHO system had glycosylation patterns more similar to antibodies produced in stable CHO cell lines than Expi293-derived antibodies. However, culture duration and temperature were found to affect protein titer, monodispersity, enzyme activity, and PTMs and should be carefully selected when using the ExpiCHO system. The ExpiCHO transient transfection systems allows for facile production of milligrams to grams of protein in CHO cells and de-risks the transition from transient to stable material during drug development. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Distinctive transforming genes in x-ray-transformed mammalian cells

    International Nuclear Information System (INIS)

    Borek, C.; Ong, A.; Mason, H.

    1987-01-01

    DNAs from hamster embryo cells and mouse C3H/10T1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and in a rat cell line, the Rat-2 cells. DNAs from unirradiated cells or irradiated and visibly untransformed cells do not produce transformed colonies. The transfectant grow in agar and form tumors in nude mice. Treatment of the DNAs with restriction endonucleases prior to transfection indicates that the same transforming gene (oncogene) is present in each of the transformed mouse cells and is the same in each of the transformed hamster cells. Southern blot analysis of 3T3 or Rat-2 transfectants carrying oncogenes from radiation-transformed C3H/10T1/2 or hamster cells indicates that the oncogenes responsible for the transformation of 3T3 cells are not the Ki-ras, Ha-ras, N-ras genes, nor are they neu, trk, raf, abl, or fms. The work demonstrates that DNAs from mammalian cells transformed into malignancy by direct exposure in vitro to radiation contain genetic sequences with detectable transforming activity in three recipient cell lines. The results provide evidence that DNA is the target of radiation carcinogenesis induced at a cellular level in vitro. The experiments indicate that malignant radiogenic transformation in vitro of hamster embryo and mouse C3H/10T1/2 cells involves the activation of unique non-ras transforming genes, which heretofore have not been described

  19. New Mammalian Expression Systems.

    Science.gov (United States)

    Zhu, Jie; Hatton, Diane

    2017-06-06

    There are an increasing number of recombinant antibodies and proteins in preclinical and clinical development for therapeutic applications. Mammalian expression systems are key to enabling the production of these molecules, and Chinese hamster ovary (CHO) cell platforms continue to be central to delivery of the stable cell lines required for large-scale production. Increasing pressure on timelines and efficiency, further innovation of molecular formats and the shift to new production systems are driving developments of these CHO cell line platforms. The availability of genome and transcriptome data coupled with advancing gene editing tools are increasing the ability to design and engineer CHO cell lines to meet these challenges. This chapter aims to give an overview of the developments in CHO expression systems and some of the associated technologies over the past few years.

  20. Evaluation of the magnetic field requirements for nanomagnetic gene transfection

    Science.gov (United States)

    Fouriki, A.; Farrow, N.; Clements, M.A.; Dobson, J.

    2010-01-01

    The objective of this work was to examine the effects of magnet distance (and by proxy, field strength) on nanomagnetic transfection efficiency. Methods non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. Results Fluorescence intensity (firefly luciferase) of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. Conclusion In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™. PMID:22110859

  1. Evaluation of the magnetic field requirements for nanomagnetic gene transfection

    Directory of Open Access Journals (Sweden)

    A. Fouriki

    2010-07-01

    Full Text Available The objective of this work was to examine the effects of magnet distance (and by proxy, field strength on nanomagnetic transfection efficiency. Methods: non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. Results: Fluorescence intensity (firefly luciferase of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. Conclusion: In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™.

  2. Modulation of microfilament protein composition by transfected cytoskeletal actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Ng, S.Y.; Erba, H.; Latter, G.; Kedes, L.; Leavitt, J.

    1988-04-01

    HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of ..beta..-actin due to coding mutations in one of two ..beta..-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional ..beta..-actin gene but not following transfection of the functional ..gamma..-actin gene. In ..gamma..-actin gene-transfected substrains that have increased rates of ..gamma..-actin synthesis, ..beta..-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both ..beta..- and ..gamma..-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal ..beta..-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.

  3. Epizone: Interlaboratory Ring Trial to Compare Dna Transfection Efficiencies

    DEFF Research Database (Denmark)

    Dory, Daniel; Albina, Emmanuel; Kwiatek, Olivier

    of viruses by reverse genetics and/or generation of mutated viruses. A large number of transfection chemicals like calcium phospate, branched organic compounds, liposomes, cationic polymers etc. are available on the market which are used by different laboratories for different cell lines. To obtain...... an overview on the efficiencies of varying transfection procedures, an interlaboratory ring trial was initiated within EPIZONE theme 5. A total of 15 participitating laboratories from 7 member institutions received RK13 cells, plasmid DNA encoding firefly luciferase under the transcriptional control...... of the human cytomegalovirus major immediate early promoter, a specially developed lysis buffer and a detailed protocol. Transfected cells were harvested in the laboratories of the participants, frozen and sent to the FLI where both the luciferase activity and protein content of the individual samples were...

  4. Turbulent Mechanical Energy Budget in Stably Stratified Baroclinic Flows over Sloping Terrain

    Science.gov (United States)

    Łobocki, Lech

    2017-09-01

    Analysis of second-moment budget equations in a slope-oriented coordinate frame exhibits the pathways of exchange between the potential energy of mean flow and the total turbulent mechanical energy. It is shown that this process is controlled by the inclination of the potential temperature gradient. Hence, this parameter should be considered in studies of turbulence in slope flows as well as the slope inclination. The concept of turbulent potential energy is generalized to include baroclinicity, and is used to explain the role of along-slope turbulent heat flux in energy conversions. A generalization of static stability criteria for baroclinic conditions is also proposed. In addition, the presence of feedback between the turbulent heat flux and the temperature variance in stably-stratified flows is identified, which implies the existence of oscillatory modes characterized by the Brunt-Väisäla frequency.

  5. HeLa-LAV, an epithelial cell line stably infected with HIV-1.

    Science.gov (United States)

    Berg, J; Doe, B; Steimer, K S; Wabl, M

    1991-01-01

    An HeLa-LAV cell line was established by infecting and subcloning previously described CD4-expressing HeLa cells with HIV-1. Cells of this line stably synthesize all major HIV proteins, release infectious particles of HIV-1, but do not die even after long term culture. More than 90% of the cells express the envelope protein gp120 on the surface. The cells can be easily and efficiently labeled with 51chromium, and exhibit a low spontaneous release. Because they are susceptible to killing by allogeneic cytotoxic T cells (CTL) when targeted to gp120, they ought to be a useful source of target cells in any kind of HIV-specific killing assays. The cells may also help studies on HIV replication in non-lymphatic/non-monocytic cells. The HeLa-LAV cell line will be freely available from the AIDS Research and Reference Reagent Program.

  6. Numerical Simulations of Stably Stratified Fluid Flow Using Compact Finite-Difference Schemes

    Science.gov (United States)

    Bodnár, T.; Fraunié, Ph.; Kozel, K.

    2010-09-01

    The aim of this paper is to present the class of high order compact schemes in the context of numerical simulation of stratified flow. The numerical schemes presented here are based on the approach outlined in Lele [1]. The numerical model presented in this contribution is based on the solution of the Boussinesq approximation by a finite-difference scheme. The numerical scheme itself follows the principle of semi-discretization, with high order compact discretization in space, while the time integration is carried out by suitable Runge-Kutta time-stepping scheme. In the case presented here the steady flow was considered and thus the artificial compressibility method was used to resolve the pressure from the modified continuity equation. The test case used to demonstrate the capabilities of the selected model consists of the flow of stably stratified fluid over low, smooth hill.

  7. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    International Nuclear Information System (INIS)

    Åmand, Helene L.; Nordén, Bengt; Fant, Kristina

    2012-01-01

    Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (“peptiplexes”) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the “chelate effect” and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.

  8. Establishment of transient and stable transfection systems for Babesia ovata.

    Science.gov (United States)

    Hakimi, Hassan; Yamagishi, Junya; Kegawa, Yuto; Kaneko, Osamu; Kawazu, Shin-Ichiro; Asada, Masahito

    2016-03-23

    Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study. In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection. After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata. The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.

  9. Efficient transfection of MG-63 osteoblasts using magnetic nanoparticles and oscillating magnetic fields.

    Science.gov (United States)

    Fouriki, A; Clements, M A; Farrow, N; Dobson, J

    2014-03-01

    To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG-63) and investigate the effects of a novel non-viral oscillating nanomagnetic gene transfection system (magnefect-nano™) in enhancing transfection efficiency (TE). MG-63 cells were transfected using MNPs coupled with a GFP-carrying plasmid. The magnefect-nano system was evaluated for transfection efficiency and potential associated effects on cell viability. MG-63 cells were efficiently transfected using MNPs and the magnefect-nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect MG-63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Cell transfection as a tool to study growth hormone action

    DEFF Research Database (Denmark)

    Norstedt, G; Enberg, B; Francis, S

    1994-01-01

    of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins...... is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance...

  11. The Effect of Environmental pH on Polymeric Transfection Efficiency

    OpenAIRE

    Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

    2011-01-01

    Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of comple...

  12. Cryopreservation of mammalian semen.

    Science.gov (United States)

    Curry, Mark R

    2007-01-01

    Mammalian spermatozoa were among the very first cells to be successfully cryopreserved and over the last five decades the use of frozen-thawed semen for artificial insemination has come to play an important role in domestic livestock production. More recently, semen freezing has increasingly been utilized in the establishment of genetic resource banks for endangered species. Semen is collected, most commonly either by use of an artificial vagina or by electroejaculation of an anaesthetized animal, and basic sperm parameters assessed. Semen is extended using a TEST-egg yolk-glycerol diluent, packaged in 0.25-mL plastic straws and slowly cooled to 5 degrees C over a period of 1-2 h. Cooled straws are frozen by suspending within liquid nitrogen vapor above the liquid nitrogen surface before plunging into the liquid phase. Straws are thawed briefly in air before immersing in a 35 degrees C water bath for 15 s, and often are used directly for insemination without any further processing.

  13. Mammalian gut immunity

    Directory of Open Access Journals (Sweden)

    Benoit Chassaing

    2014-10-01

    Full Text Available The mammalian intestinal tract is the largest immune organ in the body and comprises cells from non-hemopoietic (epithelia, Paneth cells, goblet cells and hemopoietic (macrophages, dendritic cells, T-cells origin, and is also a dwelling for trillions of microbes collectively known as the microbiota. The homeostasis of this large microbial biomass is prerequisite to maintain host health by maximizing beneficial symbiotic relationships and minimizing the risks of living in such close proximity. Both microbiota and host immune system communicate with each other to mutually maintain homeostasis in what could be called a "love-hate relationship." Further, the host innate and adaptive immune arms of the immune system cooperate and compensate each other to maintain the equilibrium of a highly complex gut ecosystem in a stable and stringent fashion. Any imbalance due to innate or adaptive immune deficiency or aberrant immune response may lead to dysbiosis and low-grade to robust gut inflammation, finally resulting in metabolic diseases.

  14. Mammalian Gut Immunity

    Science.gov (United States)

    Chassaing, Benoit; Kumar, Manish; Baker, Mark T.; Singh, Vishal; Vijay-Kumar, Matam

    2016-01-01

    The mammalian intestinal tract is the largest immune organ in the body and comprises cells from non-hemopoietic (epithelia, Paneth cells, goblet cells) and hemopoietic (macrophages, dendritic cells, T-cells) origin, and is also a dwelling for trillions of microbes collectively known as the microbiota. The homeostasis of this large microbial biomass is prerequisite to maintain host health by maximizing beneficial symbiotic relationships and minimizing the risks of living in such close proximity. Both microbiota and host immune system communicate with each other to mutually maintain homeostasis in what could be called a “love–hate relationship.” Further, the host innate and adaptive immune arms of the immune system cooperate and compensate each other to maintain the equilibrium of a highly complex gut ecosystem in a stable and stringent fashion. Any imbalance due to innate or adaptive immune deficiency or aberrant immune response may lead to dysbiosis and low-grade to robust gut inflammation, finally resulting in metabolic diseases. PMID:25163502

  15. Mammalian cell biology

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Progress is reported on studies of the molecular biology and functional changes in cultured mammalian cells following exposure to x radiation, uv radiation, fission neutrons, or various chemical environmental pollutants alone or in combinations. Emphasis was placed on the separate and combined effects of polycyclic aromatic hydrocarbons released during combustion of fossil fuels and ionizing and nonionizing radiations. Sun lamps, which emit a continuous spectrum of near ultraviolet light of 290 nm to 315 nm were used for studies of predictive cell killing due to sunlight. Results showed that exposure to uv light (254 nm) may not be adequate to predict effects produced by sunlight. Data are included from studies on single-strand breaks and repair in DNA of cultured hamster cells exposed to uv or nearultraviolet light. The possible interactions of the polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)-anthracene (DmBA) alone or combined with exposure to x radiation, uv radiation (254 nm) or near ultraviolet simulating sunlight were compared for effects on cell survival

  16. mammalian brain system

    Directory of Open Access Journals (Sweden)

    Alan Kania

    2014-06-01

    Full Text Available Relaxin-3, a member of the relaxin peptide family, was discovered in 2001 as a homologue of relaxin – a well-known reproductive hormone. However, it is the brain which turned out to be a major expression site of this newly discovered peptide. Both its molecular structure and expression pattern were shown to be very conserved among vertebrates. Extensive research carried out since the discovery of relaxin-3 contributed to the significant progress in our knowledge regarding this neuropeptide. The endogenous relaxin-3 receptor (RXFP3 was identified and the anatomy of the yet uncharacterized mammalian brain system was described, with nucleus incertus as the main center of relaxin-3 expression. Not only its diffusive projections throughout the whole brain, which reach various brain structures such as the hippocampus, septum, intergeniculate leaflet or amygdala, but also functional studies of the relaxin-3/RXFP3 signaling system, allowed this brain network to be classified as one of the ascending nonspecific brain systems. Thus far, research depicts the connection of relaxin-3 with phenomena such as feeding behavior, spatial memory, sleep/wake cycle or modulation of pituitary gland hormone secretion. Responsiveness of relaxin-3 neurons to stress factors and the strong orexigenic effect exerted by this peptide suggest its participation in modulation of feeding by stress, in particular of the chronic type. The discovery of relaxin-3 opened a new research field which will contribute to our better understanding of the neurobiological basis of feeding disorders.

  17. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Science.gov (United States)

    Yu, Dawei; Zhang, Shoufeng; Du, Weihua; Zhang, Jinxia; Fan, Zongxing; Hao, Haisheng; Liu, Yan; Zhao, Xueming; Qin, Tong; Zhu, Huabin

    2014-01-01

    Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  18. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Directory of Open Access Journals (Sweden)

    Dawei Yu

    Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  19. Localization of two mammalian cyclin dependent kinases during mammalian meiosis

    NARCIS (Netherlands)

    Ashley, T.; Walpita, D.; de rooij, D. G.

    2001-01-01

    Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids)

  20. Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies

    NARCIS (Netherlands)

    Koster, Janet; Waterham, Hans R.

    2017-01-01

    Functional studies with primary human skin fibroblasts from patients with a peroxisomal disorder often require efficient transfection with plasmids to correct the genetic defect or to express heterologous reporter proteins. Here, we describe a protocol we commonly use for efficient nonviral

  1. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  2. Optimization of in vitro culture and transfection condition of bovine ...

    African Journals Online (AJOL)

    The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase ...

  3. D-Glucosamine Promotes Transfection Efficiency during Electroporation

    Directory of Open Access Journals (Sweden)

    Kazunari Igawa

    2014-01-01

    Full Text Available D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP in the cultured cells (osteoblasts; NOS-1 cells. The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

  4. Mammalian sexual dimorphism.

    Science.gov (United States)

    McPherson, F J; Chenoweth, P J

    2012-04-01

    Sexual dimorphisms (SDs) have evolved in mammals to assure greater reproductive success for individuals, usually males. Secondary sexual characteristics (SSC) developed to further this objective, tending to be more pronounced in species which are polygynous, diurnal and open-habitat dwellers. Sexual selection has underpinned many of these changes, which are not necessarily advantageous for individual survival. Domestication has affected certain characteristics, more in terms of their quantitative rather than qualitative expression. However, restrictions imposed by domestication can also affect behaviors such as isolation and post-natal bonding while artificial selection can, by focusing on certain traits, cause unforeseen effects in genetically linked traits, which, when sex-specific or sex-linked, can be reflected in SD. On a global scale, environmental changes can have important phylogenetic implications for species which rely upon environmental cues for activities as migration, hibernation and breeding, especially when SD occurs in response to such cues. Understanding the evolutionary rationale behind the development of SDs, as well as the dynamics which occur at the interface between natural and artificial selection, allows positive insights into areas as diverse as wildlife preservation and livestock management. For both, greatest "success" should be achieved when artificial selection occurs in harmony with natural selection within a supportive environment. Thus the aim of this review is to discuss current knowledge relating to the evolution, benefits and costs of mammalian sexual dimorphisms and, where possible, draw conclusions that might be beneficial for the husbandry and propagation of mammals today. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

    Directory of Open Access Journals (Sweden)

    Cecilia González

    Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

  6. A Case Study of Offshore Advection of Boundary Layer Rolls over a Stably Stratified Sea Surface

    Directory of Open Access Journals (Sweden)

    Nina Svensson

    2017-01-01

    Full Text Available Streaky structures of narrow (8-9 km high wind belts have been observed from SAR images above the Baltic Sea during stably stratified conditions with offshore winds from the southern parts of Sweden. Case studies using the WRF model and in situ aircraft observations indicate that the streaks originate from boundary layer rolls generated over the convective air above Swedish mainland, also supported by visual satellite images showing the typical signature cloud streets. The simulations indicate that the rolls are advected and maintained at least 30–80 km off the coast, in agreement with the streaks observed by the SAR images. During evening when the convective conditions over land diminish, the streaky structures over the sea are still seen in the horizontal wind field; however, the vertical component is close to zero. Thus advected feature from a land surface can affect the wind field considerably for long times and over large areas in coastal regions. Although boundary layer rolls are a well-studied feature, no previous study has presented results concerning their persistence during situations with advection to a strongly stratified boundary layer. Such conditions are commonly encountered during spring in coastal regions at high latitudes.

  7. Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

    Science.gov (United States)

    Tan, Miao; Zhong, Sihua; Wang, Wenjie; Shen, Wenzhong

    2017-08-01

    We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H) solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i) the work function of the transparent conductive oxide layer, (ii) the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si) interface, (iii) the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H) layer, and (iv) the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT) counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

  8. Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

    Directory of Open Access Journals (Sweden)

    Miao Tan

    2017-08-01

    Full Text Available We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i the work function of the transparent conductive oxide layer, (ii the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si interface, (iii the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H layer, and (iv the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

  9. The turbulent decay of trailing vortex pairs in stably stratified environments

    Energy Technology Data Exchange (ETDEWEB)

    Holzaepfel, F.; Gerz, T.; Baumann, R.

    2000-03-01

    The decay of trailing vortex pairs in thermally stably stratified environments is investigated by means of large eddy simulations. Results of in-situ measurements in the wakes of different aircraft are used to find appropriate intitializations for the simulation of wake turbulence in the quiescent atmosphere. Furthermore, cases with weak atmospheric turbulence are investigated. It is shown that the early development of the vortices is not affected by turbulence and develops almost identically as in 2D simulations. In a quiescent atmosphere the subsequent vortex decay is controlled by the interaction of short-wave disturbances, owing to the aircraft induced turbulence, and baroclinic vorticity, owing to stable stratification. As a consequence, vertical vorticity streaks between the vortices are induced which are substantially intensified by vortex stretching and finally lead to rapid turbulent wake-vortex decay. When in addition also atmospheric turbulence is present, the long-wave instability is dominantly promoted. For very strong stratification (Fr < 1) it is observed that wake vortices may rebound but lose most of their strength before reaching the flight level. Finally, the simulation results are compared to the predictive capabilities of Greene's approximate model. (orig.)

  10. Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

    Czech Academy of Sciences Publication Activity Database

    Nejepínská, Jana; Malík, Radek; Wagner, Susan; Svoboda, Petr

    2014-01-01

    Roč. 9, č. 1 (2014), e87517 E-ISSN 1932-6203 R&D Projects: GA ČR GA204/09/0085; GA ČR(CZ) GBP305/12/G034 Grant - others:EMBO(DE) 1483 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : DOUBLE-STRANDED-RNA * INITIATION FACTOR-II * PROTEIN-KINASE PKR * GENE-EXPRESSION * MOUSE OOCYTES * MESSENGER-RNAS * HAIRPIN RNA * PHOSPHORYLATION * TRANSCRIPTION * INTERFERENCE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  11. Exploring polyamine regulation by nascent histamine in a human-transfected cell model.

    Science.gov (United States)

    Abrighach, H; Fajardo, I; Sánchez-Jiménez, F; Urdiales, J L

    2010-02-01

    There are multiple lines of evidence suggesting interplay between histamine and polyamines in several mammalian cell types. However, the complex metabolic context makes it difficult to elucidate the mechanisms involved. Histamine's effects can be elicited after its binding to any of the four subtypes of G-protein coupled histamine membrane receptors. In addition, intracellular histamine can also interfere with polyamine metabolism, since there are several metabolic connections between the synthesis and degradation pathways of both types of amines. In order to dissect the metabolic effects of intracellular histamine on polyamine metabolism, we chose a well-known cell culture line, i.e., the human embryonic kidney 293 cells (HEK-293 cells). Initially, we show that HEK-293 cells lack a polyamine metabolic response to extracellular histamine, even over a wide range of histamine concentrations. HEK-293 cells were transfected with active and inactive versions of human histidine decarboxylase, and changes in many of the overlapping metabolic factors and limiting steps were tested. Overall, the results indicate a regulatory effect of histamine on the post-transcriptional expression of ornithine decarboxylase and suggest that this effect is primarily responsible for the decrease in polyamine synthesis and partial blockade of cell-cycle progression, which should affect cell proliferation rate.

  12. Simulation of micro/nano electroporation for cell transfection

    Science.gov (United States)

    Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

    2018-03-01

    The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

  13. Deep sequencing reveals complex spurious transcription from transiently transfected plasmids

    Czech Academy of Sciences Publication Activity Database

    Nejepínská, Jana; Malík, Radek; Moravec, Martin

    2012-01-01

    Roč. 7, č. 8 (2012), e43283 E-ISSN 1932-6203 R&D Projects: GA ČR GA204/09/0085 Grant - others:EMBO(XE) 0001488 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : transient plasmid transfection * deep sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

  14. Transient transfection and expression of firefly luciferase in Giardia lamblia.

    OpenAIRE

    Yee, J; Nash, T E

    1995-01-01

    We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly lucife...

  15. Uptake of DNA by cancer cells without a transfection reagent.

    Science.gov (United States)

    Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

    2017-01-21

    Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting

  16. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    Energy Technology Data Exchange (ETDEWEB)

    Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  17. Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53

    International Nuclear Information System (INIS)

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-01-01

    The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival. (author)

  18. DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure.

    Science.gov (United States)

    Liu, Ying; Yan, Jing; Santangelo, Philip J; Prausnitz, Mark R

    2016-07-28

    Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. PUMA gene transfection can enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells.

    Science.gov (United States)

    Sun, C-G; Zhuang, J; Teng, W-J; Wang, Z; Du, S-S

    2015-05-29

    We explored whether p53 upregulated modulator of apoptosis (PUMA) gene transfection could enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells. The liposome-mediated recombinant eukaryotic expression vector PU-MA-pCDNA3 and empty vector plasmid were stably transfected into MCF-7 cells. Epirubicin (0.01-100 μM) was applied to MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells for 72 h. The MTT assay was used to calculate the cell survival rate in each group, and the 50% inhibitory concentration (IC50) was calculated. The IC50 values of epirubicin in MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were 13 ± 1.4, 1.8 ± 0.2, and 10.7 ± 1.3 μM, respectively. The sensitivity of MCF-7/PUMA cells to epirubicin increased 7.2-fold. Epirubicin induced apoptosis in MCF-7 cells dose-dependently, but MCF-7/PUMA cell-induced apoptosis was more significant compared to controls. Low concentrations of epirubicin (0.1 μM) caused low levels of apoptosis of MCF-7/pCDNA3 (1.15 ± 0.26%) and MCF-7 cells (0.9 ± 0.24%), but significantly induced apoptosis of MCF-7/PUMA cells (6.44 ± 1.46%). High epirubicin concentration (1 μM) induced apoptosis in each group, but the epirubicin MCF-7/PUMA apoptosis rate (35.47 ± 9.36%) was significantly higher than that of MCF-7 (12.6 ± 3.73%) and MCF-7/ pCDNA3 (15.2 ± 5.17%) cells (P PUMA protein expression in MCF-7/PUMA cells was significantly higher than that in MCF-7 and MCF-7/pCDNA3 cells by Western blot analysis. There-fore, stable transfection of PUMA can significantly enhance epirubicin-induced apoptosis sensitivity of MCF-7 breast cancer cells.

  20. Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

    Science.gov (United States)

    Subramanian, Mahendran; Lim, Jenson; Dobson, Jon

    2013-01-01

    Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells. PMID:23936108

  1. Turbulent jet erosion of a stably stratified gas layer in a nuclear reactor test containment

    Energy Technology Data Exchange (ETDEWEB)

    Ishay, Liel [Department of Mechanical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Bieder, Ulrich [Commissariat à l’énergie atomique et aux énergies alternatives, Centre de SACLAY DEN/SAC/DANS/DM2S/STMF/LMSF, F-91191 Gif-sur-Yvette (France); Ziskind, Gennady [Department of Mechanical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Rashkovan, Alex, E-mail: rashbgu@gmail.com [Physics Department, Nuclear Research Center Negev (NRCN), PO Box 9001, Beer-Sheva 84190 (Israel)

    2015-10-15

    Highlights: • We model stably stratified layer erosion by vertical turbulent round jet. • Separate effect studies are performed as a platform for choosing modeling approach. • A test performed in MISTRA facility, CEA, Saclay is modeled using Fluent and Trio-U codes. • The proposed modeling approach showed good agreement with the MISTRA facility LOWMA-3 test. - Abstract: A number of integral and separate effect experiments were performed in the last two decades for validation of containment computational tools. The main goal of these benchmark experiments was to assess the ability of turbulence models and computational fluid dynamics codes to predict hydrogen concentration distribution and steam condensation rate in a nuclear reactor containment in the course of severe accidents. It appears from the published literature that the predictive capability of the existing computational tools still needs to be improved. This work examines numerically the temporal evolution of helium concentration in the experiment called LOWMA-3, performed in the MISTRA facility of CEA-Saclay, France. In the experiment, helium is used to mimic hydrogen of a real-case accident. The aim of this separate effect experiment, where steam condensation was not involved, is to predict helium concentration field. The conditions of the experiment are such that both the momentum transport and molecular diffusion contributions to the mixing process are of the same order of magnitude (Fr ∼ 1). A commercial CFD code, Fluent, and a CEA in-house code, Trio-U, are used for flow and helium concentration fields temporal evolution prediction in the present study. The preliminary separate effect studies provide guidance to an optimal modeling approach for the LOWMA-3 experiment. Temporal evolution of helium concentration in the stratification layer is shown, and a comparison to the experiment is discussed. It is shown that correct modeling of the round jet flowfield is essential for a reliable

  2. Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.

    Science.gov (United States)

    Tailor, Jignesh; Kittappa, Raja; Leto, Ketty; Gates, Monte; Borel, Melodie; Paulsen, Ole; Spitzer, Sonia; Karadottir, Ragnhildur Thora; Rossi, Ferdinando; Falk, Anna; Smith, Austin

    2013-07-24

    Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

  3. Direct numerical simulations of stably-stratified sheared turbulence: Implications for oceanic mixing

    Science.gov (United States)

    Itsweire, E. C.; Holt, S. E.; Koseff, J. R.; Ferziger, J. H.

    1990-01-01

    Direct numerical simulations of the time evolution of homogeneous stably stratified turbulent shear flows have been performed for several Richardson numbers Ri and Reynolds numbers R(sub lambda) in earlier works. The results show excellent agreement with length scale models developed from laboratory experiments to characterize oceanic turbulence. When the Richardson number Ri is less than the stationary value Ri(sub s), the turbulence intensity grows at all scales, and the growth rate appears to be a function of Ri. The size of the vertical density inversions also increases. On the other hand, when Ri is greater than or equal to Ri(sub s) the largest turbulent eddies become vertically constrained by buoyancy when the Ellison (turbulence) scale L(sub E) and the Ozmidov (buoyancy) scale L(sub O) are equal. At this point, the mixing efficiency is maximal and corresponds to a flux Richardson number R(sub f) = 0.20. The vertical mass flux becomes counter-gradient when epsilon = 19(nu)N(exp 2) and vertical density overturns are suppressed in less than half a Brunt-Vaisala period. The results of the simulations were also recast in terms of the Hydrodynamic Phase Diagram introduced in fossil turbulence models. The so-called point of fossilization occurs when epsilon = 4DCN(exp 2); Gibson proposed 13DCN(exp 2). This value is in agreement with indirect laboratory observations and field observations. Finally, the validity of the steady-state models to estimate vertical eddy diffusivities in the oceanic thermocline is discussed.

  4. Turbulent jet erosion of a stably stratified gas layer in a nuclear reactor test containment

    International Nuclear Information System (INIS)

    Ishay, Liel; Bieder, Ulrich; Ziskind, Gennady; Rashkovan, Alex

    2015-01-01

    Highlights: • We model stably stratified layer erosion by vertical turbulent round jet. • Separate effect studies are performed as a platform for choosing modeling approach. • A test performed in MISTRA facility, CEA, Saclay is modeled using Fluent and Trio-U codes. • The proposed modeling approach showed good agreement with the MISTRA facility LOWMA-3 test. - Abstract: A number of integral and separate effect experiments were performed in the last two decades for validation of containment computational tools. The main goal of these benchmark experiments was to assess the ability of turbulence models and computational fluid dynamics codes to predict hydrogen concentration distribution and steam condensation rate in a nuclear reactor containment in the course of severe accidents. It appears from the published literature that the predictive capability of the existing computational tools still needs to be improved. This work examines numerically the temporal evolution of helium concentration in the experiment called LOWMA-3, performed in the MISTRA facility of CEA-Saclay, France. In the experiment, helium is used to mimic hydrogen of a real-case accident. The aim of this separate effect experiment, where steam condensation was not involved, is to predict helium concentration field. The conditions of the experiment are such that both the momentum transport and molecular diffusion contributions to the mixing process are of the same order of magnitude (Fr ∼ 1). A commercial CFD code, Fluent, and a CEA in-house code, Trio-U, are used for flow and helium concentration fields temporal evolution prediction in the present study. The preliminary separate effect studies provide guidance to an optimal modeling approach for the LOWMA-3 experiment. Temporal evolution of helium concentration in the stratification layer is shown, and a comparison to the experiment is discussed. It is shown that correct modeling of the round jet flowfield is essential for a reliable

  5. Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

    KAUST Repository

    Kumar, Abhishek

    2017-01-11

    In this paper, we characterise the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence, restricted in a vertical plane, under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) coexists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate k−3 scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a k−3 power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano–Obukhov scaling that transitions from a shallow k−11/5 form at large scales, to a steeper approximate k−3 scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterised by an approximate k−1.64 scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a k−2.5 scaling, while PE exhibits a forward transfer and its spectrum shows an approximate k−1.6 power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indicies are well explained by accounting for the scale-dependent nature of the corresponding flux.

  6. Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

    Science.gov (United States)

    Park, Eulsoon; Cho, Hong-Baek; Takimoto, Koichi

    2015-05-01

    Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    Science.gov (United States)

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Chromatin remodeling in mammalian embryos.

    Science.gov (United States)

    Cabot, Birgit; Cabot, Ryan A

    2018-03-01

    The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g., histone methylation and acetylation) and non-covalent remodeling (chromatin remodeling via remodeling complexes; e.g., SWI/SNF-mediated chromatin remodeling). Comparisons are also drawn between remodeling events that occur in embryos from a variety of mammalian species. © 2018 Society for Reproduction and Fertility.

  9. Comparison of [18F]FHBG and [14C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection

    International Nuclear Information System (INIS)

    Min, Jung-Jun; Iyer, Meera; Gambhir, Sanjiv S.

    2003-01-01

    Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells - stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [ 18 F]FHBG, [ 3 H]PCV, and [ 14 C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [ 14 C]FIAU accumulation was greater than that of [ 3 H]PCV and [ 18 F]FHBG in control cells and in HSV1-tk stably transfected cells (P 8 pfu), [ 18 F]FHBG and [ 3 H]PCV accumulation was significantly greater than that of [ 14 C]FIAU (P 18 F]FHBG and [ 3 H]PCV accumulated to a significantly greater extent than [ 14 C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P 14 C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [ 18 F]FHBG (P 14 C]FIAU, [ 18 F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [ 18 F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A-stb-tk+). [ 14 C]FIAU accumulated in HSV1-tk stably expressing cells to a greater extent than either [ 3 H]PCV or [ 18 F]FHBG. However, the accumulation of [ 3 H]PCV and [ 18 F]FHBG in adenoviral infected C6 cells or hepatocytes was equivalent to or greater than that of [ 14 C

  10. Identifying stably expressed housekeeping genes in the endometrium of fertile women, women with recurrent implantation failure and recurrent miscarriages

    OpenAIRE

    Stocker, Linden; Cagampang, Felino; Cheong, Ying

    2017-01-01

    Housekeeping genes (HKG) are presumed to be constitutively expressed throughout tissue types but recent studies have shown they vary with pathophysiology. Often, validation of appropriate HKG is not made. There is no consensus on which HKGs are most stably expressed in endometrial tissue so this study aimed to identify the most stable HKG in the endometrium of women with recurrent implantation failure (RIF) and recurrent miscarriages (RM). Inclusion criteria were women between 25-45 years (...

  11. Evolutionary dynamics of mammalian karyotypes

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2012-12-01

    Full Text Available This special volume of Cytogenetic and Genome Research (edited by Roscoe Stanyon, University of Florence and Alexander Graphodatsky, Siberian division of the Russian Academy of Sciences is dedicated to the fascinating long search of the forces behind the evolutionary dynamics of mammalian karyotypes, revealed after the hypotonic miracle of the 1950s....

  12. Technology of mammalian cell encapsulation

    NARCIS (Netherlands)

    Uludag, H; De Vos, P; Tresco, PA

    2000-01-01

    Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates

  13. PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

    Science.gov (United States)

    Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

    2015-09-14

    The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

  14. Graphene and carbon nanotube nanocomposite for gene transfection.

    Science.gov (United States)

    Hollanda, L M; Lobo, A O; Lancellotti, M; Berni, E; Corat, E J; Zanin, H

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. The Effect of Environmental pH on Polymeric Transfection Efficiency

    Science.gov (United States)

    Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

    2011-01-01

    Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6~7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1~2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. PMID:22130563

  16. Immune monitoring using mRNA-transfected dendritic cells

    DEFF Research Database (Denmark)

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by m...... and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA....

  17. Establishing malaria parasite transfection technology in South Africa.

    CSIR Research Space (South Africa)

    Van Brummelen, AC

    2010-01-01

    Full Text Available stream_source_info van Brummelen_2010.pdf.txt stream_content_type text/plain stream_size 3034 Content-Encoding UTF-8 stream_name van Brummelen_2010.pdf.txt Content-Type text/plain; charset=UTF-8 Oral ( ) / Poster (X...@csir.co.za Keywords: transfection, malaria, Plasmodium Topic: Genomics Biochemistry and Molecular Biology The most important contributing factor to the current malaria crisis is the rapid spread of parasite resistance to available anti-malarial drugs. Anti...

  18. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    Science.gov (United States)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  19. Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimization electrode configuration

    Science.gov (United States)

    Jinno, Masafumi; Tachibana, Kunihide; Motomura, Hideki; Saeki, Noboru; Satoh, Susumu

    2016-07-01

    Plasma gene transfection is expected as a safe and useful method of gene transfection. However, in this method, there is difficulty in keeping both high transfection efficiency and less cell damage simultaneously. The authors have evaluated transfection efficiency and cell viability using four different plasma sources, such as arc discharge, plasma jet, dielectric barrier discharge (DBD), and microplasma. A high transfection efficiency was achieved by discharge forms in which the electric current flows via the cells. This suggested that an electric current plays an important role in plasma gene transfection. The total volume of gas flow must be small or zero and the area in which the cells are directly irradiated by plasma must be small in order to achieve a higher cell viability. The microplasma that satisfies these conditions achieved both the highest transfection efficiency and the highest cell viability simultaneously.

  20. Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.

    Science.gov (United States)

    Woods, Georgia; Zito, Karen

    2008-02-13

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

  1. Molecular studies of fibroblasts transfected with hepatitis B virus DNA

    International Nuclear Information System (INIS)

    Chen, M.L.; Hood, A.; Thung, S.N.; Gerber, M.A.

    1987-01-01

    Two subclones (D7 and F8) derived from an NIH 3T3 mouse fibroblast cell line after transfection with hepatitis B virus (HBV) genomes, secreted significantly different amounts of HBsAg and HBeAg. DNA extracted from the subclones revealed only integrated and no extrachromosomal HBV DNA sequences as determined by the Southern blot technique with a /sup 32/P-labeled full length HBV DNA probe. The amount and integration sites of HBV sequences were significantly different in the two subclones. HBV DNA sequences coding for HBsAg and HBcAg were detected by alkaline phosphatase-conjugated, single-stranded synthetic gene-specific oligonucleotide probes revealing a larger number of copies in D7 DNA than in F8 DNA. Using a biotinylated probe for in situ hybridization, HBV DNA was found in the nuclei of all D7 cells with predominant localization to a single chromsome, but only in 10-20% of F8 cells. These observations demonstrate different integration patterns of HBV and DNA in two subclones derived from a transfected cell line and suggest that the amount of integrated HBV DNA is proportional to the amount of HBV antigens produced

  2. Towards optical cell transfection inside a micro flow cell

    Science.gov (United States)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-03-01

    For optical transfection, cells are shortly subjected to intense, focused laser radiation which leads to a temporary opening in the cell membrane. Although the method is very efficient and ensures high cell viability, the targeting of single cells with laser pulses is a tedious and slow approach. We present first measurements aiming at an experimental setup which is suitable for high throughput and automated optical cell transfection. In our setup, cells flow through a micro flow cell where they are spatially confined. The laser radiation is focused into the cell in a way that an elongated focal region is realized. This makes the time consuming aiming of the laser beam at individual cells unnecessary and opens the possibility to develop a completely automated system. The elongated laser focal region is realized by a quasi-Bessel beam which is generated by an axicon lens setup and continuously scanned from side to side of the cell. We present test measurements of the newly employed setup and discuss its suitability to be fully integrated into a flow cell sequencing system.

  3. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    Science.gov (United States)

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Exploring the ultrashort pulse laser parameter space for membrane permeabilisation in mammalian cells.

    Science.gov (United States)

    Rudhall, Andrew P; Antkowiak, Maciej; Tsampoula, Xanthi; Mazilu, Michael; Metzger, Nikolaus K; Gunn-Moore, Frank; Dholakia, Kishan

    2012-01-01

    The use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.

  5. Bioenergetics of mammalian sperm capacitation.

    Science.gov (United States)

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods.

  6. The shape of mammalian phylogeny

    DEFF Research Database (Denmark)

    Purvis, Andy; Fritz, Susanne A; Rodríguez, Jesús

    2011-01-01

    Mammalian phylogeny is far too asymmetric for all contemporaneous lineages to have had equal chances of diversifying. We consider this asymmetry or imbalance from four perspectives. First, we infer a minimal set of 'regime changes'-points at which net diversification rate has changed-identifying 15...... six simple macroevolutionary models, showing that those where speciation slows down as geographical or niche space is filled, produce more realistic phylogenies than do models involving key innovations. Lastly, an analysis of the spatial scaling of imbalance shows that the phylogeny of species within...... an assemblage, ecoregion or larger area always tends to be more unbalanced than expected from the phylogeny of species at the next more inclusive spatial scale. We conclude with a verbal model of mammalian macroevolution, which emphasizes the importance to diversification of accessing new regions...

  7. Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.

    Science.gov (United States)

    Tamm, Christoffer; Kadekar, Sandeep; Pijuan-Galitó, Sara; Annerén, Cecilia

    2016-10-01

    Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.

  8. [Optimization of triple plasmids transfection into HEK293 cells mediated by polyethylenimine].

    Science.gov (United States)

    Fu, Qiang; Li, Yan; Zheng, Zhaofen; Liu, Aizhong; Yuan, Zhenhua; Peng, Jianqiang; He, Jin

    2015-02-01

    In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.

  9. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Science.gov (United States)

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  10. Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

    Science.gov (United States)

    Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

    2013-01-01

    Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

  11. Autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity.

    Science.gov (United States)

    Xu, Tingting; Close, Dan M; Webb, James D; Ripp, Steven A; Sayler, Gary S

    2013-10-28

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

  12. Repair of ionizing radiation damage in primate αDNA transfected into rat cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.

    1992-01-01

    The time-course of repair of irradiated primate αDNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no αDNA. Plasmid pBUC4α10, containing 10 tandem 172 bp αDNA subunits in its 5kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected αDNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected αDNA in time-course experiments. αDNA continuously entered nuclei for processing in the first 2.5h after transfection. The pool of damaged bases in αDNA in NRK nuclei was detectable 2.5 h after transfection. (author)

  13. In vitro studies of magnetically enhanced transfection in COS-7 cells

    International Nuclear Information System (INIS)

    Ang, D.; Tay, C.Y.; Tan, L.P.; Preiser, P.R.; Ramanujan, R.V.

    2011-01-01

    In the magnetically enhanced gene delivery technique, DNA complexed with polymer coated aggregated magnetic nanoparticles (AMNPs) is used for effecting transfection. The aim of this study is to examine the relationship between transfection efficiency and the physical characteristics of the polymer coated AMNPs. In vitro studies of transfection efficiency in COS-7 cells were carried out using pEGFP-N1 and pMIR-REPORT complexed polyethylenimine (PEI) coated iron oxide magnetic nanoparticles. PEI coated AMNPs (PEI-AMNPs) with average individual particle diameters in the range of 8 nm to 30 nm were studied and characterized by transmission electron microscopy, vibrating sample magnetometry, X-ray diffractometry, thermal gravimetric analysis and photon correlation spectroscopy methods. PEI-A8MNP and PEI-A30MNP yielded higher transfection efficiency compared to commercial polyMAG particles as well as PEI of equivalent molar ratio of nitrogen/phosphorous (N/P ratio). The transfection efficiency was related to the physical characteristics of the PEI-AMNPs and its complexes: transfection efficiency was strongly positively correlated with saturation magnetization (Ms) and susceptibility (χ), strongly negatively correlated with N/P ratio, moderately positively correlated to zeta potential and moderately negatively correlated to hydrodynamic diameter of the complex. PEI-A8MNP and PEI-A30MNP possessing higher Ms, χ, lower N/P ratio and smaller complex size exhibited higher transfection efficiency compared to PEI-A16MNP which have weaker magnetic properties, higher N/P ratio and larger complex size. We have demonstrated that optimization of the physical properties of PEI-AMNPs is needed to maximize transfection efficiency. - Research highlights: →The transfection efficiency in magnetically enhanced gene delivery was studied. →Transfection efficiency was strongly positively correlated to magnetic properties. →Transfection efficiency was strongly negatively correlated with

  14. High-Efficient Transfection of Human Embryonic Stem Cells by Single-Cell Plating and Starvation.

    Science.gov (United States)

    Liu, Hui; Ren, Caiping; Zhu, Bin; Wang, Lei; Liu, Weidong; Shi, Jia; Lin, Jianxing; Xia, Xiaomeng; Zeng, Fei; Chen, Jiawen; Jiang, Xingjun

    2016-03-15

    Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

  15. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    Science.gov (United States)

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  16. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture

    Directory of Open Access Journals (Sweden)

    M Majumdar

    2014-01-01

    Full Text Available The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001, even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  17. Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts.

    Science.gov (United States)

    Ishiguro, Kazuhiro; Watanabe, Osamu; Nakamura, Masanao; Yamamura, Takeshi; Matsushita, Masanobu; Goto, Hidemi; Hirooka, Yoshiki

    2017-07-01

    Commercially available lipid-based transfection reagents are widely used to deliver DNA to cells. However, these lipid-based transfection reagents show poor gene transfer efficiency in primary cells. Here, we demonstrate a simple method to improve gene transfer efficiency in primary fibroblasts and hepatoblasts using a combination of lipid-based transfection reagents. Our data show that combined use of Lipofectamine LTX and FuGENE HD increases the efficiency of gene transfer compared with the use of either reagent alone, and this combination achieves the best result of any pairwise combination of Lipofectamine LTX, FuGENE HD, TransFectin, and Fibroblast Transfection Reagent.

  18. A platform for functional assessment of large variant libraries in mammalian cells

    Science.gov (United States)

    Stephany, Jason J.

    2017-01-01

    Abstract Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, promoter-less plasmids is recombined into the landing pad in each cell. Thus, every cell in the recombined pool expresses a single variant, allowing for parallel, sequencing-based assessment of variant effect. We describe a method for incorporating a single landing pad into a defined site of a cell line of interest, and show that our approach can be used generate more than 20 000 recombinant cells in a single experiment. Finally, we use our platform in combination with a sequencing-based assay to explore the N-end rule by simultaneously measuring the effects of all possible N-terminal amino acids on protein expression. PMID:28335006

  19. Evolutionary paths to mammalian cochleae.

    Science.gov (United States)

    Manley, Geoffrey A

    2012-12-01

    Evolution of the cochlea and high-frequency hearing (>20 kHz; ultrasonic to humans) in mammals has been a subject of research for many years. Recent advances in paleontological techniques, especially the use of micro-CT scans, now provide important new insights that are here reviewed. True mammals arose more than 200 million years (Ma) ago. Of these, three lineages survived into recent geological times. These animals uniquely developed three middle ear ossicles, but these ossicles were not initially freely suspended as in modern mammals. The earliest mammalian cochleae were only about 2 mm long and contained a lagena macula. In the multituberculate and monotreme mammalian lineages, the cochlea remained relatively short and did not coil, even in modern representatives. In the lineage leading to modern therians (placental and marsupial mammals), cochlear coiling did develop, but only after a period of at least 60 Ma. Even Late Jurassic mammals show only a 270 ° cochlear coil and a cochlear canal length of merely 3 mm. Comparisons of modern organisms, mammalian ancestors, and the state of the middle ear strongly suggest that high-frequency hearing (>20 kHz) was not realized until the early Cretaceous (~125 Ma). At that time, therian mammals arose and possessed a fully coiled cochlea. The evolution of modern features of the middle ear and cochlea in the many later lineages of therians was, however, a mosaic and different features arose at different times. In parallel with cochlear structural evolution, prestins in therian mammals evolved into effective components of a new motor system. Ultrasonic hearing developed quite late-the earliest bat cochleae (~60 Ma) did not show features characteristic of those of modern bats that are sensitive to high ultrasonic frequencies.

  20. Accurate and semi-automated analysis of bacterial association with mammalian cells.

    Science.gov (United States)

    Murphy, C M; Paré, S; Galea, G; Simpson, J C; Smith, S G J

    2016-03-01

    To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Mammalian cloning: advances and limitations.

    Science.gov (United States)

    Solter, D

    2000-12-01

    For many years, researchers cloning mammals experienced little success, but recent advances have led to the successful cloning of several mammalian species. However, cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not clear what technical and biological factors underlie this. Our understanding of the molecular basis of reprogramming remains extremely limited and affects experimental approaches towards increasing the success rate of cloning. Given the future practical benefits that cloning can offer, the time has come to address what should be done to resolve this problem.

  2. Processing of high-molecular-weight form adrenocorticotropin in human adrenocorticotropin-secreting tumor cell line (DMS-79) after transfection of prohormone convertase 1/3 gene.

    Science.gov (United States)

    Tateno, T; Kato, M; Tani, Y; Yoshimoto, T; Oki, Y; Hirata, Y

    2010-02-01

    Ectopic ACTH-producing tumors preferentially secrete biologically inactive ACTH precursors and ACTH-related fragments. DMS-79 is known to secrete unprocessed high-molecular-weight (HMW) form ACTH. To determine whether prohormone convertase (PC) 1/3 is involved in the abnormal processing of proopiomelanocortin (POMC), we studied whether PC1/3 and 2 genes are expressed in DMS-79, and whether overexpression of PC1/3 gene affects POMC processing pattern. Steady-state mRNA levels of PC1/3 and 2 were determined by real-time RT-PCR. Molecular weights of ACTH-related peptides were determined by chromatographical analyses coupled with ACTH and beta-endorphin (beta-END) radioimmunoassays. PC1/3 gene was transfected into DMS-79 by retrovirus transduction using pMX-IP vector encoding PC1/3 cDNA. The steady-state mRNA levels of PC1/3 and 2 in DMS-79 were lower than those in ACTH-secreting and nonfunctioning pituitary tumors. DMS-79 predominantly secreted HMW form with both ACTH and beta-END immunoreactivities by size-exclusion chromatography. After purification by immunoaffinity chromatography with anti-ACTH antibody, the apparent molecular weight of HMW form ACTH was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. After retroviral transfection of PC1/3 cDNA into DMS-79 and puromycin selection, PC1/3 stably-expressing cell line (DMS-79T) secreted two immunoreactive ACTH components, a major one coeluting with ACTH(1-39) and a minor one as a HMW form as well as two beta- END immunoreactive components coeluting with beta-lipotropic hormone and beta-END, respectively. Thus, we have established PC1/3 stably-expressing cell line (DMS-79T) capable of proteolytically processing ACTH precursor molecule(s) into mature ACTH and beta-END.

  3. A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses.

    Science.gov (United States)

    Rosa, Filipa; Sales, Kevin C; Cunha, Bernardo R; Couto, Andreia; Lopes, Marta B; Calado, Cecília R C

    2015-10-01

    Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R (2) ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R (2) = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

  4. Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

    Science.gov (United States)

    Fietz, D; Markmann, M; Lang, D; Konrad, L; Geyer, J; Kliesch, S; Chakraborty, T; Hossain, H; Bergmann, M

    2015-12-29

    Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.

  5. Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

    Science.gov (United States)

    Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

    2016-10-01

    High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

    Science.gov (United States)

    Green, David W; Kim, Eun-Jung; Jung, Han-Sung

    2015-09-01

    The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

  7. Acute and persistent infection by a transfected Mo7 strain of Babesia bovis

    Science.gov (United States)

    Stable transfection of the Mo7 strain of Babesia bovis and expression of an exogenous gene has been demonstrated in long term culture. However, the use of transfected parasites as marker vaccines or vehicles for expressing exogenous antigens in vivo requires demonstration of acute and persistent inf...

  8. Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications

    Science.gov (United States)

    King, William J.; Kouris, Nicholas A.; Choi, Siyoung; Ogle, Brenda M.; Murphy, William L.

    2012-01-01

    Non-viral transfection is a promising technique which could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density, and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density, and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105, and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity, and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications. PMID:22277991

  9. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells.

    Science.gov (United States)

    Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean; Kelesidis, Theodoros

    2017-01-01

    Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.

  10. Transfection of glioma cells with the neural-cell adhesion molecule NCAM

    DEFF Research Database (Denmark)

    Edvardsen, K; Pedersen, P H; Bjerkvig, R

    1994-01-01

    The tumor growth and the invasive capacity of a rat glioma cell line (BT4Cn) were studied after transfection with the human transmembrane 140-kDa isoform of the neural-cell adhesion molecule, NCAM. After s.c. injection, the NCAM-transfected cells showed a slower growth rate than the parent cell...

  11. [An experimental study on recombinant adenovirus p53 transfected in oral dysplastic epithelial cells].

    Science.gov (United States)

    Xu, Bo; Zhang, Song-Tao; Li, Long-Jiang; Han, Bo; Zhao, Hong-Wei; Pan, Jian

    2009-04-01

    To investigate and evaluate the appropriate virus titer and transfection efficiency of recombinant adenovirus p53 into the oral dysplastic epithelial cells (POE-9n) and provide reference for oral precancerosis research. The transfection sensitivity of adenovirus into oral dysplastic epithelial cells was evaluated by the recombinant adenovirus p53 containing green fluorescent protein (rAd-GFP). Different titre rAd -p53 was transfected into oral dysplastic epithelial cells to evaluate the effects of rAd-p53 on cell proliferation inhibition by MIT assay. The expression of exogenous p53 gene in POE-9n cells was detected by immunocytochemistry. More than 95% POE-9n cells were transfected by rAd-GFP with MOI from 100 to 500 and there was no statistical difference between different MOI values (r=-0.124, P>0.05). It was found that rAd-p53 had significant inhibition effects on POE-9n cell proliferation with MOI from 100 to 500, and there were no significant differences at 96 h and 120 h after the transfection on cell proliferation inhibition (P>0.05). P53 protein was well expressed in rAd-p53 transfected POE-9n cells. Exogenous p53 can be successfully transfected into POE-9n cells by rAd-p53 and the virus titer of MOI 100 was high enough to ensure efficient transfection.

  12. Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells

    DEFF Research Database (Denmark)

    Edvardsen, K; Brünner, N; Spang-Thomsen, M

    1993-01-01

    A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared...

  13. Nucleic acid transfection and transgenesis in parasitic nematodes.

    Science.gov (United States)

    Lok, James B

    2012-04-01

    Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins.

  14. Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering

    Science.gov (United States)

    2017-01-01

    The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

  15. Analysis of acquired resistance to cis-diamminedichloroplatinum(II) in oncogene transfected SHOK cells

    International Nuclear Information System (INIS)

    Kinashi, Yuko; Masunaga, Shinichiro; Suzuki, Minoru; Ono, Koji; Akaboshi, Mitsuhiko; Watanabe, Masami.

    1998-01-01

    SHOK (Syrian hamster Osaka-Kanazawa) cells were transfected with activated oncogenes (v-mos, c-myc, N-ras, H-ras, K-ras). These oncogene transfected cells were treated with 195m Pt-cis-diamminedichloroplatinum(II) (CDDP). Clonogenic cell survival assay showed that oncogene-transfected cells exhibited a 1.3-4.8 fold increases resistance to cisplatin compared to the parental SHOK cells. The CDDP concentration binding to DNA, RNA and protein were measured by counting the 195m Pt-radioactivity. The CDDP uptake was decreased in these oncogene transfected cells. The CDDP uptake in DNA of H-ras transfected cells decreased faster than control SHOK cells. (author)

  16. NBD-conjugated biosurfactant (MEL-A) shows a new pathway for transfection.

    Science.gov (United States)

    Ueno, Yoshinobu; Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2007-11-20

    Gene transfection is a fundamental technology for molecular and cell biology, and also clinical gene therapy. A variety of non-viral vectors have been investigated for gene transfection, but their gene delivery had remained an inefficient process. Recently, we found that a biosurfactant, mannosylerythritol lipid (MEL)-A, dramatically increased the efficiency in transfection of plasmid DNA mediated by cationic liposomes. However, its mechanism has not been understood yet. Here we examined the mechanism of the transfection mediated by cationic liposomes with NBD-conjugated MEL-A. We found that MEL-A first gradually distributed on the intracellular membranes through the plasma membranes of target cells, while the cationic liposomes with MEL-A fused to the plasma membranes in 20-35 min. Thereafter, the oligonucleotide released from the vesicles was immediately transferred to the nucleus. The present results showed a new role of non-viral vectors in transfection.

  17. The preliminary study on the inductory signal triggering the error-prone DNA repair function in mammalian cells

    International Nuclear Information System (INIS)

    Su Zaozhong; Luo Zuyu

    1989-01-01

    The nature of the signal triggering error-prone DNA repair function in mammalian cells was studied from two notions: (1) Does the inducing signal result from the direct hitting the cellular targets by DNA-damaging agents? (2) Is inhibition of DNA replication a prerequisite condition for the triggering effect? Thus, the ultraviolet (UV)-irradiated exogenous DNAs were introduced into human and rat cells by transfection. The results showed that this transfection was able to induce the error-prone repair as efficient as direct UV-irradiation to cells. Moreover, the two inductory treaetments expressed similar kinetics and dose-responses. No matter whether the introduced DNAs initiated replication, they exhibited the incuctory activity. Therefore, it can be considered that DNA lesions itself, not the direct interaction of DNA-damaging agents with specific cellular targets, serve as a triggering signal for the inductory process. Inhibition of DNA replication is not a prerequisite for the inductory signal

  18. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  19. Interaction theory of mammalian mitochondria.

    Science.gov (United States)

    Nakada, K; Inoue, K; Hayashi, J

    2001-11-09

    We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit. Copyright 2001 Academic Press.

  20. 21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

    International Nuclear Information System (INIS)

    Gaibelet, Gérald; Tercé, François; Bertrand-Michel, Justine; Allart, Sophie; Azalbert, Vincent; Lecompte, Marie-France; Collet, Xavier; Orlowski, Stéphane

    2013-01-01

    Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

  1. 21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

    Energy Technology Data Exchange (ETDEWEB)

    Gaibelet, Gérald [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France); Tercé, François [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Bertrand-Michel, Justine [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Lipidomic Platform Metatoul, Toulouse (France); Allart, Sophie [Plateau Technique d’Imagerie Cellulaire, INSERM U1043, Toulouse (France); Azalbert, Vincent [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Lecompte, Marie-France [INSERM U563, Faculté de Médecine de Rangueil, Toulouse (France); Collet, Xavier [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Orlowski, Stéphane, E-mail: stephane.orlowski@cea.fr [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France)

    2013-11-01

    Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

  2. Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency.

    Science.gov (United States)

    Silva, João P Neves; Oliveira, Ana C N; Lúcio, Marlene; Gomes, Andreia C; Coutinho, Paulo J G; Oliveira, M Elisabete C D Real

    2014-09-01

    Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results

  3. Mammalian gastrointestinal parasites in rainforest remnants

    Indian Academy of Sciences (India)

    Here, we studied the gastrointestinal parasites of nonhuman mammalian hosts living in 10 rainforest patches of the Anamalai Tiger Reserve, India. We examined 349 faecal samples of 17 mammalian species and successfully identified 24 gastroin-testinal parasite taxa including 1 protozoan, 2 trematode, 3 cestode and 18 ...

  4. Research Note Do mammalian herbivores influence invertebrate ...

    African Journals Online (AJOL)

    We investigated the indirect influence of mammalian herbivores on invertebrates, by utilising long-term mammalian herbivore exclosures in Kruger National Park. The exclosures span three distinct habitat types (crest, footslope and riparian) on a catena. By performing invertebrate collections in the exclosures and in a ...

  5. Recent integrations of mammalian Hmg retropseudogenes

    Indian Academy of Sciences (India)

    We propose that select retropseudogenes of the high mobility group nonhistone chromosomal protein genes have recently integrated into mammalian genomes on the basis of the ... [Tecle E., Zielinski L. and Kass D. H. 2006 Recent integrations of mammalian Hmg retropseudogenes. J. Genet., 85, 179–185]. Introduction.

  6. Photodynamic inactivation of mammalian viruses and bacteriophages.

    Science.gov (United States)

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  7. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    Science.gov (United States)

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  8. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    Directory of Open Access Journals (Sweden)

    Liliana Costa

    2012-06-01

    Full Text Available Photodynamic inactivation (PDI has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  9. Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina.

    Directory of Open Access Journals (Sweden)

    Satoru Moritoh

    Full Text Available The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus monkey as a "super-model" of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells, and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]. By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.

  10. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    International Nuclear Information System (INIS)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-01-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism

  11. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. (Univ. of Missouri-Columbia (USA))

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  12. Tec kinase stimulates cell survival in transfected Hek293T cells and is regulated by the anti-apoptotic growth factor IGF-I in human neutrophils.

    Science.gov (United States)

    Himpe, E; Abdul Rahim, S A; Verdood, P; Mano, H; Kooijman, R

    2013-03-01

    Previously, we showed that the phosphatidylinositol-3 kinase (PI(3)K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its down-stream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative down-stream target of PI(3)K, in neutrophils and whether this molecule is able to affect apoptosis. We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI(3)K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI(3)K activation. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with E.coli formamidopyrimidine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Mara Foresta

    Full Text Available Cigarette smoke (CS is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP. Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na(+K(+-ATPase locus (oua(r were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells' capacity to repair damaged DNA.

  14. Optimization of transfection of green fluorescent protein in pursuing mesenchymal stem cells in vivo

    Directory of Open Access Journals (Sweden)

    Pınar Elçi

    2008-12-01

    Full Text Available OBJECTIVE: Green Fluorescent Protein (GFP has been used as a marker of gene expression and a single cell marker in living organisms in cell biology studies. The important areas that GFP is used are expression levels of different genes in different organisms by inserting GFP in these genes and as a marker in living cells. In this study, we tried to optimize transfection of mesenchymal stem cells, (MSCs used for regeneration of damaged tissues in animals, by GFP containing plasmid vector by which MSCs can be followed in vivo.METHODS: To this aim, phM-GFP plasmid vector carrying GFP gene and effectene transfection reagent were used. RESULTS: The data revealed that twice transfection of MSCs resulted in higher expression of GFP for longer times as compared to once transfected MSCs. On the other hand, leaving the chemical transfection agents in the medium induced apoptosis after a while. CONCLUSION: As a conclusion we suggest the transfection of MSCs twice with 48 hours interval and removal of transfection agents after 8 hours which removed toxic and apoptotic effects of the chemicals.

  15. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    International Nuclear Information System (INIS)

    Zhao Yingzheng; Lu Cuitao

    2008-01-01

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C 3 F 8 , were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C 3 F 8 microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C 3 F 8 -filled lipid microbubbles represents an effective avenue for AS-ODNs transfer

  16. Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

    Science.gov (United States)

    Jinno, Masafumi; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu

    2016-09-01

    This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    Science.gov (United States)

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-08-17

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells.

  18. Effects of molecular size and chemical factor on plasma gene transfection

    Science.gov (United States)

    Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Jinno, Masafumi

    2016-07-01

    In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

  19. Synergistic effect of electrical and chemical factors on endocytosis in micro-discharge plasma gene transfection

    Science.gov (United States)

    Jinno, M.; Ikeda, Y.; Motomura, H.; Isozaki, Y.; Kido, Y.; Satoh, S.

    2017-06-01

    We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m-1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

  20. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells

    Science.gov (United States)

    Muir, Elizabeth M.; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W.; Keynes, Roger J.; Rogers, John H.

    2010-01-01

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury. PMID:19900493

  1. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    International Nuclear Information System (INIS)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E.; Ramos, S.G.; Silva, C.L.; Coelho-Castelo, A.A.M.

    2012-01-01

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis

  2. Preliminary study on the mechanism of radioresistance of SHG44 cells transfected by PKB

    International Nuclear Information System (INIS)

    Liu Fenju; Sun Zhiqiang; Yang Xueqin; Jiang Yaqi; Xue Jing

    2007-01-01

    Objective: To explore the radiosensitivity of SHG44 cells increased by suppressing the protein kinase B (PKB), in order to prove whether the PKB activity is related to the radioresistance of SHG44 cells. Methods: PKB gene(pCMV5.HA-m/p-PKBα(PKB), pCMV5.HA-PKBα-DD (T308D/S473D) (PKBD)) were transfected into SHG44 cells by electroporation, the cell proliferation rate was observed among the control, PKB transfected and irradiated groups by MTT assay. The laser confocal microscope was used to detect the changes of cell apoptosis and its microstructure in control, control + radiation, PKB transfected + radiation, PKBD transfected + radiation group. The proliferation of PKB transfected SHG44 cells and the relative factors of inducing apoptosis were analyzed. Results: The plasmid containing extrinsic PKB was successfully transfected into SHG44 cells and expressed PKB mRNA, while there was no expression in the control group; the proliferation rate of transfected SHG44 cells was significantly different from the control group (P 60 Co γ rays could induce SHG44 cell apoptosis with the changes of cell nuclei shape. The SHG44 cells transfected by PKB in the PKB + control group were complete, with few apoptosis cells seen, while the apoptosis was more significant in PKBD + irradiation group comparing to the control-irradiation group. Conclusions: SHG44 cells transfected by PKB could resist the cell apoptosis induced by radiation, suggesting that there were some relations between PKB activity and SHG44 cells radioresistance. (authors)

  3. Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

    Directory of Open Access Journals (Sweden)

    Min Koo Seo

    2011-02-01

    Full Text Available BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF gene in an Epstein-Barr virus (EBV-based plasmid (pEBVHGF showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF.ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

  4. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  5. Viral Cre-LoxP tools aid genome engineering in mammalian cells.

    Science.gov (United States)

    Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

    2017-01-01

    Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

  6. Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells

    DEFF Research Database (Denmark)

    Edvardsen, K; Brünner, N; Spang-Thomsen, M

    1993-01-01

    A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared...... to cells transfected with expression-vector alone or untransfected cells. However, when injected subcutaneously into nude mice, both NCAM expressing cells and control cells produced invasive tumors. Nude mice injected with NCAM positive cells developed tumors with slower growth rates as compared to those...

  7. Effect of NCAM-transfection on growth and invasion of a human cancer cell line

    DEFF Research Database (Denmark)

    Edvardsen, K; Bock, E; Jirus, S

    1997-01-01

    A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial...... basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable...

  8. Invaginating Structures in Mammalian Synapses

    Science.gov (United States)

    Petralia, Ronald S.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

    2018-01-01

    Invaginating structures at chemical synapses in the mammalian nervous system exist in presynaptic axon terminals, postsynaptic spines or dendrites, and glial processes. These invaginating structures can be divided into three categories. The first category includes slender protrusions invaginating into axonal terminals, postsynaptic spines, or glial processes. Best known examples of this category are spinules extending from postsynaptic spines into presynaptic terminals in forebrain synapses. Another example of this category are protrusions from inhibitory presynaptic terminals invaginating into postsynaptic neuronal somas. Regardless of the direction and location, the invaginating structures of the first category do not have synaptic active zones within the invagination. The second category includes postsynaptic spines invaginating into presynaptic terminals, whereas the third category includes presynaptic terminals invaginating into postsynaptic spines or dendrites. Unlike the first category, the second and third categories have active zones within the invagination. An example of the second category are mossy terminal synapses of the hippocampal CA3 region, in which enlarged spine-like structures invaginate partly or entirely into mossy terminals. An example of the third category is the neuromuscular junction (NMJ) where substantial invaginations of the presynaptic terminals invaginate into the muscle fibers. In the retina, rod and cone synapses have invaginating processes from horizontal and bipolar cells. Because horizontal cells act both as post and presynaptic structures, their invaginating processes represent both the second and third category. These invaginating structures likely play broad yet specialized roles in modulating neuronal cell signaling.

  9. Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

    DEFF Research Database (Denmark)

    Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

    2016-01-01

    Cl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and qp enhancement during TGE (post-treatment) was examined. For the TGE...

  10. [Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells].

    Science.gov (United States)

    Yin, Kai; Ma, Li; Shen, Chuan'an; Shang, Yuru; Li, Dawei; Li, Longzhu; Zhao, Dongxu; Cheng, Wenfeng

    2016-05-01

    To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture

  11. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we......-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity...

  12. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

    DEFF Research Database (Denmark)

    Khan, Aly A; Betel, Doron; Miller, Martin L

    2009-01-01

    Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition...... among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets...... of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites...

  13. DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

    Science.gov (United States)

    Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

  14. Oncogene transfection of mink lung cells: effect on growth characteristics in vitro and in vivo.

    Science.gov (United States)

    Khan, M Z; Spandidos, D A; Kerr, D J; McNicol, A M; Lang, J C; De Ridder, L; Freshney, R I

    1991-01-01

    Three sublines have been derived from the parental line Mv1Lu by transfection with normal and mutated Ha-ras, and myc oncogenes, and subsequent cloning. All the oncogenes have increased the growth rate of the cell in vitro, increased their plating efficiency in monolayer and suspension, and reduced their serum dependence. Growth in vivo as xenografts in nude mice has also been increased. Very few tumours were generated from the parental line and those that did form did so after a prolonged lag period, while the transfected lines produced tumours with 100% efficiency, and a short lag period. In general the effects of ras transfection were more extreme, with the highest growth rates and plating efficiencies in vitro and the shortest lag period and doubling times in vivo. There was no increase in plasminogen activator activity as a result of transfection, and the invasive behaviour of the lines in organotypic culture was broadly similar.

  15. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available This presentation is about the photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses. It outlines the background on embryonic stem cells (ES) and phototransfection....

  16. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    Science.gov (United States)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  17. Effect of NCAM-transfection on growth and invasion of a human cancer cell line

    DEFF Research Database (Denmark)

    Edvardsen, K; Bock, E; Jirus, S

    1997-01-01

    A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial...... basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable...... of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth...

  18. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available disease- iPS, dopaminergic neurons Transplantation • Autologous- bone marrow, tissue defects, leukemia • Haematopoietic- blood dieases, autoimmune disorders • Mesenchymal- neurological disorders Phototransfection • Transfection refers...

  19. Characterization of pseudorabies viruses produced in mammalian ...

    African Journals Online (AJOL)

    The full-length genome DNA of pseudorabies virus (PrV) was transfected into swine testis (ST) cells using calcium phosphate method to generate the infectious viruses. The resulting viruses were identified by indirect immunofluorescence and were used to infect African green monkey kidney (Vero) cells. Our results showed ...

  20. Acidity-responsive gene delivery for "superfast" nuclear translocation and transfection with high efficiency.

    Science.gov (United States)

    Zhu, Jing-Yi; Zeng, Xuan; Qin, Si-Yong; Wan, Shuang-Shuang; Jia, Hui-Zhen; Zhuo, Ren-Xi; Feng, Jun; Zhang, Xian-Zheng

    2016-03-01

    In principle, not only efficient but rapid transfection is required since it can maximize the bioavailability of vector-carried gene prior to the cellular excretion. However, the "rapid" goal has been paid few attentions so far in the research field of vector-aided transfection. As a pioneering attempt, the present study designed a lysosome-targeting acidity-responsive nanoassembly as gene vectors, which proved the amazing potency to mediate the "Superfast" transnuclear gene transport and gene transfection with high efficiency in vitro and in vivo. The nanoassembly was constructed on the pH-reversible covalent boronic acid-diol coupling between 1,3-diol-rich oligoethylenimine (OEI-EHDO) and phenylboronic acid modified cholesterol (Chol-PBA). The rapid and efficient nuclei-tropic delivery and transfection was demonstrated to highly rely on the lysosome-acidity induced assembly destruction followed by the easy liberation of gene payloads inside cells. The nanoassembly-mediated transfection at 8 h can afford the outcome even comparable to that achieved at 48 h by the golden standard of PEI25k, and the transfection efficiency can still remain at a high level during 48 h. In contrast, time-dependent efficiency enhancement was identified for the transfections using PEI25k and OEI-EHDO as delivery vectors. Moreover, owing to the hydroxyl-rich surface, this delivery nanosystem presented strong tolerance to the serum-induced transfection inhibition that frequently occurred for the polycationic gene vectors such as PEI25k. The in vitro and in vivo results manifested the low toxicity of this bio-decomposable nanoassembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. [EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Yu, Fengxu; Chen, Yongen; Chen, Feng; Xia, Jiyi; Liu, Hongduan; Fu, Yong; Li, Miaoling; Liao, Bin

    2016-04-01

    To investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. LVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3PLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94% N₂, 1% O₂, and 5% CO₂ for 0, 3, 6, 9, and 12 hours (group B1 and group C1). The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t = 17.525, P = 0.013). The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.

  2. Evaluation of the transfection efficacies of quaternary ammonium salts prepared from sophorolipids

    OpenAIRE

    Delbeke, E,; Lozach, Olivier; Le Gall, T; Berchel, Mathieu,; Montier, T,; Jaffres, Paul-Alain; Van Geem, K,; Stevens, C,

    2016-01-01

    International audience; Five quaternary ammonium amphiphilic compounds were synthesized from sophorolipid 1. These compounds were formulated in aqueous media and some of them (5 and 6) produced well-defined supra-molecular aggregates which were characterized by DLS and zeta measurements. Their capacity to transfect four different eukaryotic cell lines in vitro was assessed. To evaluate the influence of the carbohydrate head group from the sophorolipids on the transfection efficacies, their de...

  3. Bioactive polyphenols from muscadine grape and blackcurrant stably concentrated onto protein-rich matrices for topical applications.

    Science.gov (United States)

    Plundrich, N; Grace, M H; Raskin, I; Ann Lila, M

    2013-08-01

    Natural botanical agents that are antimicrobial, or that modulate skin hyperpigmentation via tyrosinase inhibition, are increasingly sought in the cosmetic industry. In this study, an efficient tactic is demonstrated for concentrating and stabilizing skin-beneficial bioactive compounds from muscadine grape and blackcurrant juice or muscadine pomace, into hemp flour (HF), hemp protein isolate (HPI) and soy protein isolate (SPI) matrices suitable for cosmetic applications. Anthocyanins were most efficiently captured from blackcurrant juice into HF (8.39 mg g(-1) ). HPI most effectively captured total phenolics from muscadine pomace (72.32 and 77.32 mg g(-1) from Noble and Carlos, respectively), while the three matrices incorporated highest levels of ellagic acid, gallic acid, and PAC B1 from Noble muscadine grape juice. The enriched matrices demonstrated effective in vitro inhibition of tyrosinase (up to 57.29% for blackcurrant juice-HPI matrix), and in general, juice sources provided greater inhibition on L-dopamine oxidation by tyrosinase than pomace sources. The polyphenol-enriched matrices effectively inhibited microbial proliferation in a screening assay against Staphylococcus aureus bacteria, whereas untreated HF, HPI or SPI did not inhibit bacterial growth. The technology of combining and stably concentrating phytoactive polyphenols with proteins has potential use for cosmetic topical applications. © 2013 John Wiley & Sons Ltd.

  4. Identifying stably expressed housekeeping genes in the endometrium of fertile women, women with recurrent implantation failure and recurrent miscarriages.

    Science.gov (United States)

    Stocker, Linden; Cagampang, Felino; Cheong, Ying

    2017-11-01

    Housekeeping genes (HKG) are presumed to be constitutively expressed throughout tissue types but recent studies have shown they vary with pathophysiology. Often, validation of appropriate HKG is not made. There is no consensus on which HKGs are most stably expressed in endometrial tissue so this study aimed to identify the most stable HKG in the endometrium of women with recurrent implantation failure (RIF) and recurrent miscarriages (RM). Inclusion criteria were women between 25-45 years (n = 45) suffering recurrent miscarriage (RM), recurrent implantation failure (RIF) or fertile controls. Endometrial biopsies were taken and total RNA extraction, cDNA synthesis and PCR was performed using 10 candidate HKG. The genes were arranged in terms of stability and normalisation was determined. Several HKGs not previously tested in endometrial samples were found to be more stable than those previously identified as the most stable. Of these, the 5 most stable HKG (in order of stability) were Prdm4 (PR domain 4) > Ube4a (Ubiquitin-Conjugating Enzyme 4a) > Enox2 (Ecto-NOX Disulfide-Thiol Exchanger 2) > Ube2d2 (Ubiquitin-conjugating enzyme E2D 2) > Actb (Actin beta). We therefore recommend using at least four of the aforementioned HKG for normalisation of endometrial tissues taken from patients with RM and RIF.

  5. Mammalian synthetic biology: emerging medical applications

    Science.gov (United States)

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  6. Spatio-temporally controlled transfection by quantitative injection into a single cell.

    Science.gov (United States)

    Kwon, Hyosung; Park, Hang-soo; Yu, Jewon; Hong, Sunghoi; Choi, Yeonho

    2015-10-01

    Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    Science.gov (United States)

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy.

    Science.gov (United States)

    Zhong, Xiao; Panus, David; Ji, Weihang; Wang, Chun

    2015-03-02

    Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.

  9. Novel mechanism of gene transfection by low-energy shock wave

    Science.gov (United States)

    Hoon Ha, Chang; Cheol Lee, Seok; Kim, Sunghyen; Chung, Jihwa; Bae, Hasuk; Kwon, Kihwan

    2015-01-01

    Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo. PMID:26243452

  10. Transfection of rat embryo cells with mutant p53 increases the intrinsic radiation resistance

    International Nuclear Information System (INIS)

    Pardo, F.S.; Su, M.; Gerweck, L.; Schmidt, E.V.; Borek, C.; Preffer, F.; Dombkowski, D.

    1994-01-01

    Dominant oncogenic sequences have been shown to modulate the intrinsic radiation sensitivity of cells of both human and murine tumor cell lines. Whether transfection with candidate tumor-suppressor genes can modulate intrinsic radiation sensitivity is unknown. The data presented here demonstrate that transfection of rat embryo cells with a mutant p53 allele can increase the intrinsic radiation resistance of cells in vitro. First, transfection with mutant p53 resulted in transformed cellular morphology. Second, the transfected clone and the corresponding pooled population of transfected clones were more resistant to ionizing radiation in vitro. Last, analyses of the parameters of cell kinetics suggested that the radiobiological effects were unlikely to be due to altered parameters of cell kinetics at the time of irradiation, suggesting that mutant p53 altered the intrinsic radiation resistance of transfected cells by a more direct mechanism. Further experimentation will be necessary to develop a mechanistic approach for the study of these alterations. 29 refs., 3 figs., 2 tabs

  11. Investigation of transfection efficacy with transcatheter arterial transporting transferring to enhance p53 gene

    International Nuclear Information System (INIS)

    Lu Qin; Niu Huanzhang; Zhu Guangyu; An Yanli; Qiu Dinghong; Teng Gaojun

    2007-01-01

    Objective: To investigate the function of transferrin-DNA complex, transported by transferrin(Tf) and trans-arterial injection via interventional approach be the duel-target-orientated delivery and the transferring into malignant cells to get more effective therapy. Methods: p53-LipofectAMINE ligand with different concentrations of Tf (0, 10, 25, 50, 100 μg)transfected the 4 strains including LM6,Hep3B,YY and L02 in vitro to evaluate the gene transfection efficiency through western blot. Then, after setting up the VX2 hepatocarcinoma models, we delivered the Tf-p53-LipofectAMlNE complex into the hepatic arteries via interventional techniques to analyse the transfection efficiency in vivo. Results: Tf, within the range of l0 100 μg, could increase gene transfection efficiency mediated by liposome, and the efficiency increases with the raise of Tf concentration. Combination with interventional technique to inject Tf-DNA complex into tumor arteries, gene transfection efficiency was enhanced in rabbit models. Conclusion: Tf can enhance gene-liposome transfection efficiency, furthermore with combination of interventional catheter technique, there would be a potential duel-target-orientated gene therapy method. (authors)

  12. Single-Tailed Lipidoids Enhance the Transfection Activity of Their Double-Tailed Counterparts.

    Science.gov (United States)

    Wu, Yihang; Li, Linxian; Chen, Qing; Su, Yi; Levkin, Pavel A; Davidson, Gary

    2016-01-11

    Cationic lipid-like molecules (lipidoids) are widely used for in vitro and in vivo gene delivery. Nearly all lipidoids developed to date employ double-tail or multiple-tail structures for transfection. Single-tail lipidoids are seldom considered for transfection as they have low efficiency in gene delivery. So far, there is no detailed study on the contribution to transfection efficiency of single-tail lipidoids when combined with standard double-tail lipidoids. Here, we use combinatorial chemistry to synthesize 17 double-tail and 17 single-tail lipidoids using thiol-yne and thiol-ene click chemistry, respectively. HEK 293T cells were used to analyze transfection efficiency by fluorescence microscopy and calculated based on the percentage of cells transfected. The size and zeta potential of liposomes and lipoplexes were characterized by dynamic light scattering (DLS). Intracellular DNA delivery and trafficking was further examined using confocal microscopy. Our study shows that combining single with double-tail lipidoids increases uptake of lipoplexes, as well as cellular transfection efficiency.

  13. Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

    Science.gov (United States)

    Gomaa, Fatma; Garcia, Paulo A; Delaney, Jennifer; Girguis, Peter R; Buie, Cullen R; Edgcomb, Virginia P

    2017-09-01

    We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  14. A study on the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles.

    Science.gov (United States)

    Li, Huiling; Chen, Jinwen; Xu, Xuan; Yang, Ruhao; Xiang, Xudong; Zhang, Dongshan

    2016-02-01

    To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (Ptransfection of CTGF-antisense-ODN into kidney. The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.

  15. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    Science.gov (United States)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  16. Exploring the Correlation Between Lipid Packaging in Lipoplexes and Their Transfection Efficacy

    Science.gov (United States)

    Moghaddam, Behfar; McNeil, Sarah E.; Zheng, Qinguo; Mohammed, Afzal R.; Perrie, Yvonne

    2011-01-01

    Whilst there is a large body of evidence looking at the design of cationic liposomes as transfection agents, correlates of formulation to function remain elusive. In this research, we investigate if lipid packaging can give further insights into transfection efficacy. DNA lipoplexes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method. Each of the formulations was prepared by hydration in dH2O or phosphate buffer saline (PBS) to investigate the effect of buffer salts on lipoplex physicochemical characteristics and in vitro transfection. In addition, Langmuir monolayer studies were performed to investigate any possible correlation between lipid packaging and liposome attributes. Using PBS, rather than dH2O, to prepare the lipoplexes increased the size of vesicles in most of formulations and resulted in variation in transfection efficacies. However, one combination of lipids (DSPE:DOTAP) could not form liposomes in PBS, whilst the DSPE:DSTAP combination could not form liposomes in either aqueous media. Monolayer studies demonstrated saturated lipid combinations offered dramatically closer molecular packing compared to the other combinations which could suggest why this lipid combination could not form vesicles. Of the lipoplexes prepared, those formulated with DSTAP showed higher transfection efficacy, however, the effect of buffer on transfection efficiency was formulation dependent. PMID:24309311

  17. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite

    KAUST Repository

    Zhang, G.

    2015-06-25

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in ‘double transfections’, in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.

  18. Isolation of a XP-A cell clone with intermediate UV sensitivity following transfection with genomic mouse DNA. Isolation and characterization of transfected sequences

    International Nuclear Information System (INIS)

    Blum, M.

    1987-04-01

    The work presented here describes an attempt to restore repair proficiency in xp cells of the most UV sensitive complementation group A by DNA mediated gene transfer. The transfection conditions employed for the cell line used here (XP12Rotk - 1) have been optimized. (orig./MG) [de

  19. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  20. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Directory of Open Access Journals (Sweden)

    Elizabeth Muir

    Full Text Available There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed

  1. The Synergistic Effect between Electrical and Chemical Factors in Plasma Gene/Molecule-Transfection

    Science.gov (United States)

    Jinno, Masafumi

    2016-09-01

    This study has been done to know what kind of factors in plasma and processes on cells promote plasma gene/molecule transfection. We have discovered a new plasma source using a microcapillary electrode which enables high transfection efficiency and high cell survivability simultaneously. However, the mechanism of the transfection by plasma was not clear. To clarify the transfection mechanisms by micro plasma, we focused on the effects of electrical (current, charge, field, etc.) and chemical (radicals, RONS, etc.) factors generated by the micro plasma and evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones. At first, the necessity of the electrical factors was estimated by the laser produced plasma (LPP). Mouse L-929 fibroblast cell was cultured on a 96-well plate or 12-well micro slide chamber. Plasmids pCX-EGFP in Tris-EDTA buffer was dropped on the cells and they were exposed to the capillary discharge plasma (CDP) or the LPP. In the case of the CDP, the plasma was generated between the tip of the capillary electrode and the cells so that both electrical and chemical factors were supplied to the cells. In this setup, about 20% of average transfection efficiency was obtained. In the case of the LPP, the plasma was generated apart from the cells so that electrical factors were not supplied to the cells. In this setup, no transfection was observed. These results show that the electrical factors are necessary for the plasma gene transfection. Next, the necessity of the chemical factors was estimated the effect of catalase to remove H2O2 in CDP. The transfection efficiency decreased to 0.4 by scavenging H2O2 with catalase. However, only the solution of H2O2 caused no gene transfection in cells. These results shows that H2O2 is important species to cause gene/molecule transfection but still needs a synergistic effect with electrical or other chemical factors. This work was partly supported by

  2. A family of cationic polyamides for in vitro and in vivo gene transfection.

    Science.gov (United States)

    Zhang, Chengnan; Jin, Rong; Zhao, Peng; Lin, Chao

    2015-08-01

    The purpose of this study is to develop biodegradable cationic polyamides for non-viral gene delivery and elucidate their structural effects on gene transfection activity. To this end, a group of novel cationic polyamides were synthesized by polycondensation reaction between different di-p-nitrophenyl esters and tertiary amine-containing primary diamines. These linear polyamides have flexible alkylene group (ethylene or propylene), protonable amino group and bioreducible disulfide linkage in the polyamide main chain. The alkylene group and disulfide linkage in these polyamides have a distinct effect on their gene delivery properties including buffering capacity, gene binding ability and intracellular gene release profile. Those cationic polyamides containing disulfide linkage and 1,4-bis(3-aminopropyl)piperazine (BAP) residue exhibited high buffering capacity (endosomal escape ability), high gene binding ability, and intracellular gene release ability, thus inducing fast gene nucleus translocation and robust gene transfection in vitro against different cell lines and rat bone marrow mesenchymal stem cells. Moreover, the transfection efficiencies in vitro were comparable or higher than those of 25 kDa branched polyethylenimine and Lipofectamine 2000 transfection agent as positive controls. These cationic polyamides and their polyplexes were of low cytotoxicity when an optimal transfection efficacy was achieved. In vivo transfection tests showed that bioreducible BAP-based polyamides were applicable for intravenous gene delivery in a mouse model, leading to higher level of transgene expression in the liver as compared to 22 kDa linear polyethylenimine as a positive control. These cationic polyamides provide a useful platform to elucidate the relationship between chemical functionalities and gene transfection activity. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. Combining polyethylenimine and Fe(III) for mediating pDNA transfection.

    Science.gov (United States)

    Jorge, Andreia F; Röder, Ruth; Kos, Petra; Dias, Rita S; Wagner, Ernst; Pais, Alberto A C C

    2015-06-01

    The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA-PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. Fe(III) emerges as a good supporting condensing agent to modulate pDNA-PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Gold nanoparticle mediated laser transfection for efficient siRNA mediated gene knock down.

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    Dag Heinemann

    Full Text Available Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

  5. Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

    Science.gov (United States)

    Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

    2013-01-01

    Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

  6. Preparation, characterization, and efficient transfection of cationic liposomes and nanomagnetic cationic liposomes

    Directory of Open Access Journals (Sweden)

    Samadikhah HR

    2011-10-01

    Full Text Available Hamid Reza Samadikhah1,*, Asia Majidi2,*, Maryam Nikkhah2, Saman Hosseinkhani11Department of Biochemistry, 2Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran *These authors contributed equally to this work Purpose: Cationic liposomes (CLs are composed of phospholipid bilayers. One of the most important applications of these particles is in drug and gene delivery. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs has some problems, including low transfection efficiency in vivo. The aim of this study was to develop novel CLs containing magnetite to overcome the deficiencies. Patients and methods: CLs and magnetic cationic liposomes (MCLs were prepared using the freeze-dried empty liposome method. Luciferase-harboring vectors (pGL3 were transferred into liposomes and the transfection efficiencies were determined by luciferase assay. Firefly luciferase is one of most popular reporter genes often used to measure the efficiency of gene transfer in vivo and in vitro. Different formulations of liposomes have been used for delivery of different kinds of gene reporters. Lipoplex (liposome–plasmid DNA complexes formation was monitored by gel retardation assay. Size and charge of lipoplexes were determined using particle size analysis. Chinese hamster ovary cells were transfected by lipoplexes (liposome-pGL3; transfection efficiency and gene expression level was evaluated by luciferase assay. Results: High transfection efficiency of plasmid by CLs and novel nanomagnetic CLs was achieved. Moreover, lipoplexes showed less cytotoxicity than polyethyleneimine and Lipofectamine™. Conclusion: Novel liposome compositions (1,2-dipalmitoyl-sn-glycero-3-phosphocholine [DPPC]/dioctadecyldimethylammonium bromide [DOAB] and DPPC/cholesterol/DOAB with high transfection efficiency can be useful in gene delivery in vitro. MCLs can also be used for targeted gene delivery, due to

  7. piggyBac transposon-mediated cellular transgenesis in mammalian forebrain by in utero electroporation.

    Science.gov (United States)

    Chen, Fuyi; Maher, Brady J; LoTurco, Joseph J

    2014-07-01

    In utero electroporation (IUE) is an effective transfection method for delivering plasmid DNA into neural progenitor cells and neurons of mammalian neocortex in vivo. Although IUE is effective at delivering multiple DNA plasmids into populations of cells, unfortunately plasmids delivered into neural progenitor cells remain largely episomal and often get inactivated or lost after cell division. This results in a form of "birthdate" labeling in which only the cell types that do not undergo a second cell division continue to express the transfected plasmids. This limits the application of IUE with standard plasmids and precludes its use in experiments where manipulating or labeling the complete cell lineage of a progenitor is desired. To circumvent this episomal loss of plasmid in IUE, we have used a binary piggyBac transposon system to induce nonviral genomic integration of transgenes. These transgenes do not appear to inactivate after cell division, and this results in stable somatic cellular transgenesis of neurons and glia. Like standard IUE, the system can be used with multiple combinations of plasmids to achieve multicolor labeling and both loss-of-function and gain-of-function manipulations. In this protocol, we describe the method for delivering a binary piggyBac transposon plasmid system by IUE. © 2014 Cold Spring Harbor Laboratory Press.

  8. Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

    Science.gov (United States)

    Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S; Ellisman, Mark H; Ting, Alice Y

    2018-01-01

    Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). PMID:28796234

  9. Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

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    Tokmakova Irina L

    2007-11-01

    Full Text Available Abstract Background RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. Results Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. Conclusion Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.

  10. Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

    Directory of Open Access Journals (Sweden)

    Zhixin Lin

    Full Text Available The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799 which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

  11. Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord.

    Science.gov (United States)

    Gwak, So-Jung; Nice, Justin; Zhang, Jeremy; Green, Benjamin; Macks, Christian; Bae, Sooneon; Webb, Ken; Lee, Jeoung Soo

    2016-04-15

    Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1%, and 8.1%, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration. Gene therapy is a promising strategy to overcome barriers to axonal regeneration in the injured central nervous system. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to cytotoxicity and low transfection efficiency in the presence of serum proteins. Here, we report cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that are capable of efficiently transfecting reporter

  12. Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

    Science.gov (United States)

    Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

    2013-10-01

    Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and

  13. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  14. Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior.

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    Daniele Pezzoli

    Full Text Available BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW branched polyethylenimine (bPEI is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%. Along the Chi-g-bPEI(x series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7% was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x copolymers. Crucially, we have demonstrated

  15. Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior.

    Science.gov (United States)

    Pezzoli, Daniele; Olimpieri, Francesca; Malloggi, Chiara; Bertini, Sabrina; Volonterio, Alessandro; Candiani, Gabriele

    2012-01-01

    Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting

  16. The development of mechanically formed stable nanobubbles intended for sonoporation-mediated gene transfection.

    Science.gov (United States)

    Abdalkader, Rodi; Kawakami, Shigeru; Unga, Johan; Higuchi, Yuriko; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2017-11-01

    In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.

  17. Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells

    Science.gov (United States)

    Kelly, Abby M; Plautz, Sarah A; Zempleni, Janos; Pannier, Angela K

    2016-01-01

    Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4–15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6–10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells. PMID:26478250

  18. Photoenhanced gene transfection by a curcumin loaded CS-g-PZLL micelle.

    Science.gov (United States)

    Lin, Jian-Tao; Pan, Wen-Jia; Zhang, Jun-Ai; Wang, Wei; Zhong, Jia; Su, Jia-Min; Li, Tong; Zou, Ying; Wang, Guan-Hai

    2017-09-01

    The codelivery of drug and gene is a promising method for cancer treatment. In our previous works, we prepared a cationic micelles based on chitosan and poly-(N-3-carbobenzyloxylysine) (CS-g-PZLL), but transfection ratio of CS-g-PZLL to Hela cell was low. Herein, to improve the transfection efficiency of CS-g-PZLL, curcumin was loaded in the CS-g-PZLL micelle. After irradiation, the obtained curcumin loaded micelle showed a better transfection, and the p53 protein expression in Hela cells was higher. The apoptosis assay showed that the complex could induce a more significant apoptosis to Hela cells than that of curcumin or p53 used alone, and the curcumin loaded micelle inducing apoptosis was best after irradiation. Therefore, CS-g-PZLL is a safe and effective carrier for the codelivery of drug/gene, and curcumin could be used as a photosensitizer to induce a photoenhanced gene transfection, which should be encouraged in improving transfection and tumor therapy. Copyright © 2017. Published by Elsevier B.V.

  19. In vivo and in vitro recombination of lambda DNA in CaC12 transfection.

    Science.gov (United States)

    Betcke, A; Pfeifer, M; Pöhlmann, C H; Kurth, M; Hartmann, M; Liebscher, D H

    1980-01-01

    Using CaCl2 mediated transfection with Lambda DNA fragments, in vitro joining by ligase and in vivo recombination with helper phage DNA are effective systems for generating artificial recombinants. Recombination efficiencies are 20--30% in the in vitro and in vivo recombination systems. At 30 to 37 degree C T4 ligase mainly joins natural cohesive alpha ends, while at 12 degrees C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector alpha 401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCl2 transfection. For in vivo recombination a new CaCl2 transfection system was developed, termed postinfection-dependent CaCl2 transfection system, which is based on the infection of recipient cells with helper phages after transfection. In marker rescue experiments using this method not only single but also double recombination occurred between two independent alpha DNA fragments and the helper phage DNA.

  20. Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

    International Nuclear Information System (INIS)

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-01-01

    Highlights: → Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-β genes both in vitro and in vivo. → Ultrasound-mediated IFN-β transfection inhibited proliferation of melanoma cells in vitro. → Ultrasound-mediated IFN-β transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  1. Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes.

    Science.gov (United States)

    Pappalardo, Juan Sebastián; Quattrocchi, Valeria; Langellotti, Cecilia; Di Giacomo, Sebastián; Gnazzo, Victoria; Olivera, Valeria; Calamante, Gabriela; Zamorano, Patricia I; Levchenko, Tatyana S; Torchilin, Vladimir P

    2009-02-20

    Antigen presenting cells (APC) are among the most important cells of the immune system since they link the innate and the adaptative immune responses, directing the type of immune response to be elicited. To modulate the immune response in immune preventing or treating therapies, gene delivery into immunocompetent cells could be used. However, APC are very resistant to transfection. To increase the efficiency of APC transfection, we have used liposome-based lipoplexes additionally modified with cell-penetrating TAT peptide (TATp) for better intracellular delivery of a model plasmid encoding for the enhanced-green fluorescent protein (pEGFP). pEGFP-bearing lipoplexes made of a mixture of PC:Chol:DOTAP (60:30:10 molar ratio) with the addition of 2% mol of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate (plain-L) or TATp-PEG-PE (TATp-L) were shown to effectively protect the incorporated DNA from degradation. Uptake assays of rhodamine-labeled lipoplexes and transfections with the EGFP reporter gene were performed with APC derived from the mouse spleen. TATp-L-based lipoplexes allowed for significantly enhanced both, the uptake and transfection in APC. Such a tool could be used for the APC transfection as a first step in immune therapy.

  2. Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lei [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Department of Physiology, Nankai University School of Medicine, Tianjin 300071 (China); Carr, Aprell L. [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Ping; Lee, Jessica; McGregor, Mary [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Lei, E-mail: Li.78@nd.edu [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States)

    2014-07-11

    Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.

  3. Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor

    DEFF Research Database (Denmark)

    Pedersen, U B; Norby, B; Jensen, Anders A.

    1994-01-01

    Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines...

  4. Enhanced transfection efficiency and reduced cytotoxicity of novel lipid-polymer hybrid nanoplexes

    DEFF Research Database (Denmark)

    Jain, Sanyog; Kumar, Sandeep; Agrawal, Ashish Kumar

    2013-01-01

    The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency...... size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher...... uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively...

  5. Tetracycline-regulated transgene expression in hippocampal neurones following transfection with adenoviral vectors.

    Science.gov (United States)

    Harding, T C; Geddes, B J; Noel, J D; Murphy, D; Uney, J B

    1997-12-01

    A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)-responsive On/Off regulatory elements into type-5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet-On and AdTet-Off systems. Following transfection with the AdTet-On system no EGFP-fluorescent cells could be detected until doxycycline was added. The AdTet-Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.

  6. Purification of DNA for the transfection of a Spodoptera frugiperda cell line.

    Science.gov (United States)

    Slack, Jeffrey M; Lawrence, Susan D

    2002-01-01

    Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.

  7. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

    Science.gov (United States)

    Zhu, Zhengrong; Puglisi, Jaime; Connors, David; Stewart, Jeremy; Herbst, John; Marino, Anthony; Sinz, Michael; O'Connell, Jonathan; Banks, Martyn; Dickinson, Kenneth; Cacace, Angela

    2007-03-01

    Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

  8. Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium.

    Science.gov (United States)

    Prow, Tarl W; Bhutto, Imran; Kim, Sahng Y; Grebe, Rhonda; Merges, Carol; McLeod, D Scott; Uno, Koichi; Mennon, Mohamed; Rodriguez, Li; Leong, Kam; Lutty, Gerard A

    2008-12-01

    Chitosan, PCEP (poly{[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium iodide] ethyl phosphate}), and magnetic nanoparticles (MNPs) were evaluated for the safe delivery of genes in the eye. Rabbits were injected with nanoparticles either intravitreally (IV) or subretinally (SR) and sacrificed 7 days later. Eyes were grossly evaluated for retinal pigment epithelium abnormalities, retinal degeneration, and inflammation. All eyes were cryopreserved and sectioned for analysis of toxicity and expression of either enhanced green or red fluorescent proteins. All of the nanoparticles were able to transfect cells in vitro and in vivo. IV chitosan showed inflammation in 12/13 eyes, whereas IV PCEP and IV MNPs were not inflammatory and did not induce retinal pathology. SR PCEP was nontoxic in the majority of cases but yielded poor transfection, whereas SR MNPs were nontoxic and yielded good transfection. Therefore, we conclude that the best nanoparticle evaluated in vivo was the least toxic nanoparticle tested, the MNP.

  9. Involvement of opsins in mammalian sperm thermotaxis

    Science.gov (United States)

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-01-01

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways—the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors. PMID:26537127

  10. DNA repair in non-mammalian animals

    International Nuclear Information System (INIS)

    Mitani, Hiroshi

    1984-01-01

    Studies on DNA repair have been performed using microorganisms such as Escherichia coli and cultured human and mammalian cells. However, it is well known that cultured organic cells differ from each other in many respects, although DNA repair is an extremely fundamental function of organisms to protect genetic information from environmental mutagens such as radiation and 0 radicals developing in the living body. To answer the question of how DNA repair is different between the animal species, current studies on DNA repair of cultured vertebrate cells using the methods similar to those in mammalian experiments are reviewed. (Namekawa, K.)

  11. Better smelling through genetics: mammalian odor perception.

    Science.gov (United States)

    Keller, Andreas; Vosshall, Leslie B

    2008-08-01

    The increasing availability of genomic and genetic tools to study olfaction-the sense of smell-has brought important new insights into how this chemosensory modality functions in different species. Newly sequenced mammalian genomes-from platypus to dog-have made it possible to infer how smell has evolved to suit the needs of a given species and how variation within a species may affect individual olfactory perception. This review will focus on recent advances in the genetics and genomics of mammalian smell, with a primary focus on rodents and humans.

  12. A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells

    DEFF Research Database (Denmark)

    Pedersen, Charlotte Demuth; Fang, J J; Pedersen, Anders Elm

    2009-01-01

    Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation...... with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show...... that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface...

  13. Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

    Science.gov (United States)

    Misra, Santosh K.; Biswas, Joydeep; Kondaiah, Paturu; Bhattacharya, Santanu

    2013-01-01

    Background Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies. PMID:23861884

  14. Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

    International Nuclear Information System (INIS)

    Takamatsu, Shinji; Furukawa, Takako; Mori, Tetsuya; Yonekura, Yoshiharu; Fujibayashi, Yasuhisa

    2005-01-01

    The transplantation of functional cells such as dopaminergic cells into damaged tissue is now clinically ongoing, but at present the population of surviving cells at the transplantation site mostly cannot be noninvasively examined. To visualize surviving transplanted functional cells using a noninvasive method, we chose the estrogen receptor ligand binding domain (ERL) as a reporter molecule and 16α-[ 18 F]-fluoro-17β-estradiol (FES) for its ligand. We used a mouse embryonic stem (ES) cell line for recipient cells as a model. To obtain ES cells that constitutively or inducibly express ERL, we transfected two types of expression vectors into EB5 parental ES cell line using the lipofection method and obtained about 30 clones for each of the two types of transfectants. Then, to examine the expression level of ERL, we performed Western blotting analysis. Ligand uptake experiments were carried out using [ 3 H]-estradiol with or without excessive unlabeled estradiol for control cells and ERL transfectants. Each selected clone was also used for in vivo positron emission tomography (PET) imaging studies involving FES in nude mice transplanted with control cells and ERL transfectants. In some of the clones transfected with the inducible-type ERL gene, protein was expressed much higher than in the controls. However, constitutive-type ERL gene-transfected ES cells showed no protein production in spite of their gene expression activity being considerably high. All clones also expressed equal levels of the Oct-3/4 gene, a marker of pluripotency, in comparison with the parental cells. Also, the specific uptake of [ 3 H]-estradiol was over 30 times higher in inducer-treated ERL-expressing ES cells compared to untreated control cells. Finally, by performing dynamic PET imaging, we successfully visualized ERL-expressing teratomas using FES

  15. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-03-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to /sup 32/P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C ..-->.. C x G or G x C ..-->.. T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria.

  16. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  17. Intracellular characterization of Gag VLP production by transient transfection of HEK 293 cells.

    Science.gov (United States)

    Cervera, Laura; González-Domínguez, Irene; Segura, María Mercedes; Gòdia, Francesc

    2017-11-01

    Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 10 10 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet

  18. In vivo molecular imaging and radionuclide (131I) therapy of human nasopharyngeal carcinoma cells transfected with a lentivirus expressing sodium iodide symporter.

    Science.gov (United States)

    Shi, Shuo; Zhang, Min; Guo, Rui; Miao, Ying; Hu, Jiajia; Xi, Yun; Li, Biao

    2015-01-01

    Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using 125I and 131I therapy in vivo. We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; 125I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with 131I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors. qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more 125I than CNE-2Z cells and xenografts. In vitro, 131I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, 131I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of 131I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67. Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.

  19. Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production.

    Science.gov (United States)

    Xia, YuXiu C; Sun, ShanShan; Kuek, Li Eon; Lopata, Andreas L; Hulett, Mark D; Mackay, Graham A

    2011-08-01

    Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca(2+) or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38(MAPK) and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Enhancer evolution across 20 mammalian species

    DEFF Research Database (Denmark)

    Villar, Diego; Berthelot, Camille; Aldridge, Sarah

    2015-01-01

    by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements...

  1. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R

    2014-03-26

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  2. endogenous retrovirus sequences expressed in male mammalian

    African Journals Online (AJOL)

    2002-01-02

    Jan 2, 2002 ... the human genome. Because of such hypotheses, in this communication, we discuss the findings of various studies that have demonstrated expression of endogenous retrovirus-like particles in male mammalian reproductive tissues. In addition, we discuss the biological implications of the presence of these ...

  3. Architecture of mammalian respiratory complex I.

    Science.gov (United States)

    Vinothkumar, Kutti R; Zhu, Jiapeng; Hirst, Judy

    2014-11-06

    Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.

  4. Locomotor circuits in the mammalian spinal cord

    DEFF Research Database (Denmark)

    Kiehn, Ole

    2006-01-01

    Intrinsic spinal networks, known as central pattern generators (CPGs), control the timing and pattern of the muscle activity underlying locomotion in mammals. This review discusses new advances in understanding the mammalian CPGs with a focus on experiments that address the overall network...... approaches that have the potential to elucidate the function of populations of CPG interneurons are also discussed....

  5. Endogenous retrovirus sequences expressed in male mammalian ...

    African Journals Online (AJOL)

    Objectives: To review the research findings on the expression of endogenous retroviruses and retroviral-related particles in male mammalian reproductive tissues, and to discuss their possible role in normal cellular events and association with disease conditions in male reproductive tissues. Data sources: Published ...

  6. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...

  7. Transfection of myoblasts in primary culture with isomeric cationic cholesterol derivatives

    NARCIS (Netherlands)

    Bischoff, Rainer; Cordier, Y.; Perraud, F.; Thioudellet, C.; Braun, S.; Pavirani, A.

    1997-01-01

    Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three

  8. Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform.

    Science.gov (United States)

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2016-09-01

    Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Transfection of tumor-infiltrating T cells with mRNA encoding CXCR2

    DEFF Research Database (Denmark)

    Idorn, Manja; thor Straten, Eivind Per; Svane, Inge Marie

    2016-01-01

    infused T cells migrating to the tumor and the clinical response, but also that only a small fraction of adoptively transferred Tcells reach the tumor site. In this chapter, we describe a protocol for transfection of TILs with mRNA encoding the chemokine receptor CXCR2 transiently redirecting...

  10. Comparative Analysis of Non-viral Transfection Methods in Mouse Embryonic Fibroblast Cells.

    Science.gov (United States)

    Lee, Migi; Chea, Kathleen; Pyda, Rajyalakshmi; Chua, Melissa; Dominguez, Isabel

    2017-07-01

    Mouse embryonic fibroblast (MEF) cells are an important in vitro model for developmental biology, disease, and reprogramming studies. However, as with other primary cells, they are challenging to transfect. Although viral gene-delivery methods achieve high gene-delivery efficiency, challenges with cell mutagenesis and safety among others have led to the use and improvement of non-viral gene-delivery methods in MEF cells. Despite the importance of gene delivery in MEF cells, there is limited comparison of method/reagent efficacy. In this study, we compared the effectiveness of different gene-delivery methods and several reagents currently available in MEF cells by introducing a plasmid containing enhanced green fluorescent protein (EGFP). We analyze transfection efficiency by EGFP fluorescence. Our results suggest that two gene-delivery methods-electroporation and magnetofection in combination with a lipid reagent, are the most efficient transfection methods in MEF cells. This study provides a foundation for the selection of transfection methods or reagents when using MEF cells.

  11. Development of a confocal ultrasound device using an inertial cavitation control for transfection in-vitro

    Science.gov (United States)

    Mestas, J. L.; Chettab, K.; Roux, S.; Prieur, F.; Lafond, M.; Dumontet, C.; Lafon, C.

    2015-12-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. We developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (peGFP- C1) in adherent and non-adherent cell lines. The frequency spectrum of the signal receive by a hydrophone is used to compute a cavitation index (CI) representative of the inertial cavitation activity. The influence of the CI on transfection efficiency, as well as reproducibility were determined. A real-time feedback loop control on CI was integrated in the process to regulate the cavitation level during sonoporation. In both adherent and non-adherent cell lines, the sonoporation device produced a highly efficient transfection of peGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. Moreover, the sonoporation of non-adherent cell lines Jurkat and K562 was found to be equivalent to nucleofection in terms of efficiency and toxicity while these two cell lines were resistant to transfection with lipofection.

  12. Efficient Transfection of siRNA by Peptide Dendrimer-Lipid Conjugates.

    Science.gov (United States)

    Kwok, Albert; Eggimann, Gabriela A; Heitz, Marc; Reymond, Jean-Louis; Hollfelder, Florian; Darbre, Tamis

    2016-12-02

    Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure-activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure-activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    Science.gov (United States)

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-04-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

  14. Transfection mediated by gemini surfactants : Engineered escape from the endosomal compartment

    NARCIS (Netherlands)

    Bell, PC; Bergsma, M; Dolbnya, IP; Bras, W; Stuart, MCA; Rowan, AE; Feiters, MC; Engberts, JBFN

    2003-01-01

    The structure of the lipoplex formed from DNA and the sugar-based cationic gemini surfactant 1, which exhibits excellent transfection efficiency, has been investigated in the pH range 8.8-3.0 utilizing small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-TEM). Uniquely, three

  15. Enhanced gene transfection by photochemical internalization of protomine sulfate/DNA complexes

    Science.gov (United States)

    Hirschberg, Henry; Mathews, Marlon B.; Shih, En-Chung; Madsen, Steen J.; Kwon, Young Jik

    2012-02-01

    Introduction: One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of the GFP indicator gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells. Materials and Methods: U251 monolayers were incubated in AlPcS2a for 18 h. The monolayers were incubated with non-viral vectors for either 4 or 18 hrs. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched PEI or protomine sulfate (PS), were used with the plasmid construct (GFP-PTEN). Results: PS was much less toxic to the gliomas cells compared to BPEI but was highly inefficient at gene transfection. PCI resulted in a 5-10 fold increase in GFP protein expression compared to controls. Conclusions: Collectively, the results suggest that AlPcS2a-mediated PCI can be used to enhance transfection of tumor suppressor genes in glioma cells.

  16. Characterization of a tachykinin peptide NK sub 2 receptor transfected into murine fibroblast B82 cells

    Energy Technology Data Exchange (ETDEWEB)

    Van Giersbergen, P.L.M. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States) Univ. of Cincinnati, OH (United States)); Shatzer, S.A.; Buck, S.H. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States)); Henderson, A.K.; Lai, J.; Yamamura, Henry, I. (Univ. of Arizona, Tucson (United States)); Nakanishi, Shigetada (Kyoto Univ. (Japan))

    1991-03-01

    Membranes isolated from a murine fibroblast B82 cell line (SKLKB82{number sign}3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of (His({sup 125}I){sup 1})neurokinin A ({sup 125}I-NKA) to a single population of sites with a B{sub max} of 147 fmol/mg of protein and a K{sub d} of 0.59 nM. The ligand binding in SKLKB82{number sign}3 cells was reversible. Thus, SKLKB82{number sign}3 cells have been transfected with NK{sub 2} receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK{sub 2} receptors, NK{sub 2} receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of {sup 125}I-NKA binding by nucleotide analogues was markedly different in SKLKB82{number sign}3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK{sub 2} receptors.

  17. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  18. Elevation of Transfection Efficiency by Conjugation of Poly(amindoamine)-diethylenetriamine (PAM-DET) with Dexamethasone

    International Nuclear Information System (INIS)

    Jeong, Yunseong; Park, Jihye; Jin, Geunwoo; Park, Jongsang

    2012-01-01

    We successfully conjugated hydrophobic group, dexamethasone onto the surface of PAM-DET to synthesize PAM-DET-DX to form polyplexes with enhanced stability against ionic strength. We evaluated its stability by measuring the size of its polyplexes; the conjugated PAM-DET polyplex showed decreased growth compared to the PAM-DET polyplex in an environment with increased ionic strength, which implies that the conjugated PAM-DET has enhanced stability against increased ionic strength. Furthermore, conjugation of hydrophobic group caused a slight increase in the transfection efficiency without inducing toxicity. Of course, it isn't a neglectable factor that nuclear localization effect of DX can drive the advanced transfection efficiency of PAM-DET-DX polyplex. It means that the hydrophobic moieties which have some other positive properties in transfection are good candidates that can be introduced to non-viral polymeric gene delivery carrier. This strongly indicates that the introduction of hydrophobic moiety on PAM-DET is a good method to enhance polyplex stability against ionic strength without diminishing its advantageous properties, such as high transfection efficiency and low cytotoxicity

  19. Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA

    NARCIS (Netherlands)

    Olbrich, C; Bakowsky, U; Muller, RH; Kneuer, C

    2001-01-01

    The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1

  20. Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency.

    Science.gov (United States)

    Wang, Bing; Zhao, Rui-Mo; Zhang, Ji; Liu, Yan-Hong; Huang, Zheng; Yu, Qing-Ying; Yu, Xiao-Qi

    2017-08-18

    Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids containing linkages with different rigidity were designed and synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These lipids could give much higher transfection efficiency than those containing more flexible aliphatic linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve such problem and further increase the transfection efficiency, some lysosomotropic reagents were used to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofectamine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application. This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection abilities of cationic lipids and affords information to construct safe and efficient gene vector towards practical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

    Science.gov (United States)

    Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

    2016-02-01

    Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

  2. GENE TRANSFER ON Betta imbellis THROUGH TRANSFECTION METHOD WITH DIFFERENT DNA CONCENTRATION

    Directory of Open Access Journals (Sweden)

    Eni Kusrini

    2016-12-01

    Full Text Available Big size betta (Giant have a high economic value compared to normal size betta, and over expression of growth hormone gene can produce giant fish.  As an initial step of giant transgenic betta productions, this study was conducted in order to obtain DNA plasmid concentration which provide higher hatching and survival rate of betta larvae.  Construction of PhGH pCcBA gene contains growth hormone gene of Siamese catfish (PhGH and it is controlled by the CCBA promoter. Betta imbellis broodstocks were spawned naturally, and embryos were collected 1-2 minutes after spawning time. One hundred embryos were dipped in 2 mL of transfectan X-treme gene which containp CcBA-PhGH construction genes (50 µg/mL, on room temperature for about 30 minutes. Treatments on this study were different transfectant : DNA plasmid ratiosnamely:A (0,75 µL: 0,25 µL; B (0,75 µL : 0,50 µL; C (0,75 µL: 0,75 µL, D as Control 1(without transfectant, 0,25 µL DNA; E.as Control 2(0,75 µL transfectant, without DNA, and Fas control 3 (without transfectant and without DNA. Every treatments was repeated three times.  Transfection embryos were hatched on a container (1L Volume. Study results showed that hatching rate and larvae survival rate  (4 days after hatching on treatment A were the same with the control, but slightly higher than B and C treatments. PCR analysis with DNA template showing that PhGH gene were found on embryos and larvae (pooled sample of treatment A, B and C. Furthermore, RT-PCR analysis showing the existence of mRNA PhGH expression on embryos and larvae (pooled sample. Therefore, embryo transfection with transfectant ratio 0,75 µL and  DNA 0,25 µLshowing the best results.

  3. Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

    Science.gov (United States)

    Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

    2017-11-01

    Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    Directory of Open Access Journals (Sweden)

    Morral Núria

    2011-01-01

    Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  5. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  6. Marked transfection enhancement by the DPL (DNA/peptide/lipid) complex.

    Science.gov (United States)

    Moon, Ik-Jae; Kang, Hyungu; Seu, Young-Bae; Chang, Byeong-Churl; Song, Dae-Kyu; Park, Jong-Gu

    2007-10-01

    A short peptide, corresponding to the nuclear localization signal of the human immunodeficiency virus-1 Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was modified by adding a cysteine residue at the COOH terminus. The peptide was mixed with a reporter plasmid, and then with cationic lipids, to form a tripartite complex, DNA/peptide/lipid (DPL). Various cell lines were treated with the DPL complex and compared for transfection efficiency with those of the conventional DNA/lipid (DL) complex. With the simple inclusion of the peptide, the DPL complex showed much enhanced transfection. Meanwhile, the plasmid DNA mixed only with the peptide exhibited some improvement but with much lower transfection than the DPL complex. When the DPL complex was formed with various cationic lipids, the DOSPA/DOPE exhibited superior transfection efficiency than the other cationic lipids tested at the optimal ratio of 1:3:5 (w:w:w) in many cell types. At the optimal ratio of the DPL components, transfection efficiency was routinely shown to be approximately 10-fold higher for reporter gene expression than that of the conventional DL complex. Furthermore, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with antisense oligos, k-ras-RiAS, delivered as a DPL complex, tumor growth was markedly suppressed. This study shows that the DPL complex, which is easy to formulate by ordered mixing, can be employed for a much enhanced cellular uptake of a transgene both in vitro and in vivo.

  7. Inclusion of the helper lipid dioleoyl-phosphatidylethanolamine in solid lipid nanoparticles inhibits their transfection efficiency.

    Science.gov (United States)

    de Jesus, Marcelo B; Radaic, Allan; Hinrichs, Wouter L J; Ferreira, Carmen V; de Paula, Eneida; Hoekstra, Dick; Zuhorn, Inge S

    2014-02-01

    Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of helper lipids on the transfection efficiency with cationic liposomes, the effect of the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of approximately 150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000' (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1-2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective 'packaging' of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.

  8. Gene therapy of transplant arteriopathy by liposome-mediated transfection of endothelial nitric oxide synthase.

    Science.gov (United States)

    Iwata, A; Sai, S; Moore, M; Nyhuis, J; de Fries-Hallstrand, R; Quetingco, G C; Allen, M D

    2000-11-01

    Transplant arteriopathy is the major factor limiting long-term survival after cardiac transplantation. We have previously demonstrated that liposome-mediated gene delivery of endothelial nitric oxide synthase (eNOS) to donor hearts reduces ischemia-reperfusion injury by blocking NFkappaB activation, adhesion molecule expression, and leukocyte infiltration. In this study, we used gene transfer of eNOS in a rabbit carotid transplant model to see whether these same effects would similarly ameliorate transplant arteriopathy. Liposomes complexed to the gene encoding eNOS were injected into donor carotid arterial segments that were transplanted orthotopically into recipient carotid arteries (n = 10). Controls included transplanted carotids transfected with liposomes complexed to empty plasmids (no functional gene) (n = 4) and transplanted carotids treated with saline (n = 6). Transplanted arteries were harvested for processing at 21 days. Intima/media (I/M) area ratios were calculated by computerized image analysis. Infiltrating T-lymphocytes and macrophages, and expression of VCAM-1 and ICAM-1 were quantified on immunocytochemistry. The I/M ratio was significantly reduced in eNOS-transfected arteries compared with arteries transfected with empty plasmids and saline-treated controls. Compared to transplanted control arteries, eNOS-transfected arteries demonstrated significantly reduced T-cell infiltration into the intima and significantly reduced macrophage infiltration into the media. Cell surface expression of VCAM-1 and ICAM-1 were both reduced in eNOS-transfected arteries. ENOS gene delivery can suppress neointimal lesion formation and T-lymphocyte and macrophage infiltration in transplanted arteries, associated with a reduction in relevant adhesion molecule expression. Thus, gene therapy with eNOS may not only reduce ischemia-reperfusion injury but may also ameliorate transplant arteriopathy in transplanted hearts.

  9. Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.

    Science.gov (United States)

    Nooh, Mohammed M; Bahouth, Suleiman W

    2017-01-01

    G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β 1 -adrenergic receptor (β 1 -AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β 1 -AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review. © 2017 Elsevier Inc. All rights reserved.

  10. Effect on radiosensitivity in human umbilical vein endothelial cells after transfection of pcDNA3.1 + Apel plasmid

    International Nuclear Information System (INIS)

    Tan Yonghong; Xiang Debing; Shi Xikai; Yin Xiaoling; Wang Dong

    2008-01-01

    Objective: To investigate the possible effects on radiosensitivity in human umbilical vein endothelial cells after transfection of pcDNA3.1 + Apel plasmid. Methods: The expressing vector pcDNA3.1 + Apel, the control vector pcDNA3.1 + or non-transfection cells was irradiated by 2, 4, 6, and 8 Gy photon beam at 48 h post-transfection. The value of initial and residual Oliver tail moment (OTM) under the alkaline single cell gelelectrophoresis assay and the colony forming test were utilized as the markers for the evaluation of cells intrinsic radiosensitivity. The effect on radiosensitivity in human umbilical vein endothelial cells after transfection of the expressing vector pcDNA3.1 + Apel was analyzed according to the radio-dose, compared to the empty vecor control and non-transfection cells. Results: The initial and residual OTM value of endothelial cells transfected by 3 μg pcDNA3.1 + Apel plasmid was lower significantly than ones of endothelial cells untransfected at 2 Gy irradiation (P 0.05), and SF 2 was higher remarkably in transfected cells than one in untransfected cells (P 4 , SF 6 and SF 8 were no significant differences (all of P>0.05). Conclusions: The transfection of pcDNA3.1 + Apel plasmid could enhance radioresistance of endothelial cells to the low-dose irradiation. (authors)

  11. Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches.

    Directory of Open Access Journals (Sweden)

    Li Xia

    Full Text Available Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs and 17% of outer hair cells (OHCs were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively was slightly higher, but the difference was not significant.

  12. Improvement of AdCMV-GFP gene transfection efficiency induced by heavy-ion beam irradiation on murine melanoma cells

    International Nuclear Information System (INIS)

    Duan Xin; Min Fengling; Liu Bing; Zhou Qingming; Li Xiaoda; Wang Yanling; Chinese Academy of Sciences, Beijing; Zhang Hong; Qiu Rong; Hao Jifang; Zhou Guangming; Gao Qingxiang

    2007-01-01

    The effect of 12 C 6+ beam irradiation on AdCMV-GFP (a replication deficient recombinant adenoviral vector containing CMV promoter and green fluorescent protein) gene transfection efficiency for murine melanoma cell B16 has been investigated. B16 cells infected with AdCMV-GFP were irradiated by different doses of 12 C 6+ beam. The transfection efficiency was assessed by flow cytometry (FCM). Results show that 12 C 6+ beam irradiation can improve transfection efficiency of AdCMV-GFP on murine melanoma cell B16 in a dose-dependent manner. In addition, the transfection efficiency in pre-tranfection plus irradiation group is higher than that in pre-irradiation plus transfection group at the same dose irradiation dose. (authors)

  13. Mammalian Sperm Motility: Observation and Theory

    KAUST Repository

    Gaffney, E.A.

    2011-01-21

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics. © 2011 by Annual Reviews. All rights reserved.

  14. Scaling up the curvature of mammalian metabolism

    Directory of Open Access Journals (Sweden)

    Juan eBueno

    2014-10-01

    Full Text Available A curvilinear relationship between mammalian metabolic rate and body size on a log-log scale has been adopted in lieu of thelongstanding concept of a 3/4 allometric relationship (Kolokotrones et al. 2010. The central tenet of Metabolic Ecology (ME states that metabolism at the individual level scales-up to drive the ecology of populations, communities and ecosystems. If this tenet is correct, the curvature of metabolism should be perceived in other ecological traits. By analyzing the size scaling allometry of eight different mammalian traits including basal and field metabolic rate, offspring biomass production, ingestion rate, costs of locomotion, life span, population growth rate and population density we show that the curvature affects most ecological rates and

  15. Antibody transfection into neurons as a tool to study disease pathogenesis.

    Science.gov (United States)

    Douglas, Joshua N; Gardner, Lidia A; Lee, Sangmin; Shin, Yoojin; Groover, Chassidy J; Levin, Michael C

    2012-09-26

    Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP). How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1). Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis. Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and

  16. Structure and function of mammalian cilia

    DEFF Research Database (Denmark)

    Satir, Peter; Christensen, Søren T

    2008-01-01

    In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number...... of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease....

  17. Mammalian evolution may not be strictly bifurcating.

    Science.gov (United States)

    Hallström, Björn M; Janke, Axel

    2010-12-01

    The massive amount of genomic sequence data that is now available for analyzing evolutionary relationships among 31 placental mammals reduces the stochastic error in phylogenetic analyses to virtually zero. One would expect that this would make it possible to finally resolve controversial branches in the placental mammalian tree. We analyzed a 2,863,797 nucleotide-long alignment (3,364 genes) from 31 placental mammals for reconstructing their evolution. Most placental mammalian relationships were resolved, and a consensus of their evolution is emerging. However, certain branches remain difficult or virtually impossible to resolve. These branches are characterized by short divergence times in the order of 1-4 million years. Computer simulations based on parameters from the real data show that as little as about 12,500 amino acid sites could be sufficient to confidently resolve short branches as old as about 90 million years ago (Ma). Thus, the amount of sequence data should no longer be a limiting factor in resolving the relationships among placental mammals. The timing of the early radiation of placental mammals coincides with a period of climate warming some 100-80 Ma and with continental fragmentation. These global processes may have triggered the rapid diversification of placental mammals. However, the rapid radiations of certain mammalian groups complicate phylogenetic analyses, possibly due to incomplete lineage sorting and introgression. These speciation-related processes led to a mosaic genome and conflicting phylogenetic signals. Split network methods are ideal for visualizing these problematic branches and can therefore depict data conflict and possibly the true evolutionary history better than strictly bifurcating trees. Given the timing of tectonics, of placental mammalian divergences, and the fossil record, a Laurasian rather than Gondwanan origin of placental mammals seems the most parsimonious explanation.

  18. Radiation effects in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Hill, C.K.; Han, A.; Elkind, M.M.; Wells, R.L.; Buess, E.M.; Lin, C.M.

    1985-01-01

    The purpose of this research effort is to elucidate the mechanisms for the radiation-induced changes in mammalian cells that lead to cell death, mutation, neoplastic transformation, DNA damage, and chromosomal alterations. Of particular interest are the effects of low-dose-rate and fractionated irradiation on these end points with respect to the mechanisms whereby these effects are influenced by cellular repair processes, inhibitors, and promoters that act at the genetic or biochemical level. 17 refs

  19. Glia in mammalian development and disease

    OpenAIRE

    Zuchero, J. Bradley; Barres, Ben A.

    2015-01-01

    Glia account for more than half of the cells in the mammalian nervous system, and the past few decades have witnessed a flood of studies that detail novel functions for glia in nervous system development, plasticity and disease. Here, and in the accompanying poster, we review the origins of glia and discuss their diverse roles during development, in the adult nervous system and in the context of disease.

  20. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  1. Mammalian Synthetic Biology: Engineering Biological Systems.

    Science.gov (United States)

    Black, Joshua B; Perez-Pinera, Pablo; Gersbach, Charles A

    2017-06-21

    The programming of new functions into mammalian cells has tremendous application in research and medicine. Continued improvements in the capacity to sequence and synthesize DNA have rapidly increased our understanding of mechanisms of gene function and regulation on a genome-wide scale and have expanded the set of genetic components available for programming cell biology. The invention of new research tools, including targetable DNA-binding systems such as CRISPR/Cas9 and sensor-actuator devices that can recognize and respond to diverse chemical, mechanical, and optical inputs, has enabled precise control of complex cellular behaviors at unprecedented spatial and temporal resolution. These tools have been critical for the expansion of synthetic biology techniques from prokaryotic and lower eukaryotic hosts to mammalian systems. Recent progress in the development of genome and epigenome editing tools and in the engineering of designer cells with programmable genetic circuits is expanding approaches to prevent, diagnose, and treat disease and to establish personalized theranostic strategies for next-generation medicines. This review summarizes the development of these enabling technologies and their application to transforming mammalian synthetic biology into a distinct field in research and medicine.

  2. Comparison of amphibian and mammalian thyroperoxidase ...

    Science.gov (United States)

    Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high con

  3. Evolution of the mammalian lysozyme gene family

    Science.gov (United States)

    2011-01-01

    Background Lysozyme c (chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known. Results Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution. Conclusions The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties. PMID:21676251

  4. Evolution of the mammalian lysozyme gene family

    Directory of Open Access Journals (Sweden)

    Biegel Jason M

    2011-06-01

    Full Text Available Abstract Background Lysozyme c (chicken-type lysozyme has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known. Results Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution. Conclusions The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.

  5. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    Energy Technology Data Exchange (ETDEWEB)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-02-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

  6. Optimization of transfection conditions and analysis of siRNA potency using real-time PCR.

    Science.gov (United States)

    Cheng, Angie; Magdaleno, Susan; Vlassov, Alexander V

    2011-01-01

    RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.

  7. [Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

    Science.gov (United States)

    Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

    2008-06-01

    To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

  8. Developing a puncture-free in ovo chicken transfection strategy based on bypassing albumen nucleases.

    Science.gov (United States)

    Amini, Hamid-Reza; Pakdel, Abbas; Shahr-Babak, Hossein Moradi; Eghbalsaied, Shahin

    2017-03-15

    Chicken is a dual-purpose animal important from both agricultural and medical aspects. Even though significant improvements have been made in chicken transgenesis technologies, chicken genome manipulation has not been widely used in developmental biology. This study was aimed to evaluate chicken egg white nuclease properties and thereof plausibility of devising an in vivo transfection technology without causing physical damage to the embryo. First, the nuclease activity of egg albumen was assessed. The egg white nucleases were strongly active in degrading DNA and RNA. The egg white DNase activity was comparable to commercially available DNase-I. Nuclease activities were also assessed after heating, proteinase K, or EDTA treatment. Unlike proteinase K, both heating and EDTA were noticeably effective for the nuclease inactivation. Simultaneous application of lipoplex form of DNA (1 μg pDB2: 3 μl Lipofectamine2000) and EDTA showed a synergistic effect in protection against egg white nucleases. Finally, we injected the lipoplexes with or without EDTA close to the embryo at day0, but outside the embryonic epiblast. Implementation of a scrutinized PCR assay indicated that transfection took place only when EDTA was complemented to the lipoplexes. The transfection rate of day4 embryos and the hatched chicks were 54.5 and 30.0%, respectively. EGFP expression was detected in two out of three transgenic chicks. In conclusion, this study provided a detail analysis of chicken egg albumen nuclease properties and suggested the feasibility of developing a puncture-free handmade technology for transfection of the chicken embryo. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes.

    Science.gov (United States)

    Kim, Bieong-Kil; Hwang, Guen-Bae; Seu, Young-Bae; Choi, Jong-Soo; Jin, Kyeong Sik; Doh, Kyung-Oh

    2015-10-01

    The effects of lipid compositions on their physicochemical properties and transfection efficiencies were investigated. Four liposome formulations with different 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) to dioleoylphosphatidylethanolamine (DOPE) weight ratios were investigated, that is, weight ratios 1:0 (T1P0), 3:1 (T3P1), 1:1 (T1P1), and 1:3 (T1P3). Mean sizes of liposomes were influenced by their lipid composition and the preparation concentration at the time of sonication. Zeta potentials of liposomes were inversely correlated with their liposome sizes. However, neither liposome sizes nor zeta potentials were correlated with transfection efficiency. The optimum composition of liposomes was cell-line dependent (T1P0 and T3P1 for Huh7 and AGS, T3P1 and T1P1 for COS7, and T1P1 and T1P3 for A549). The shape of lipoplexes was changed from lamellar to inverted hexagonal structure according to the increased ratio of DOPE, but there was no definite advantage of specific structure in transfection efficiency throughout all used cell lines. However, cellular internalization was consistently faster in T1P0, T3P1, T1P1 compared to T1P3 in all cell lines, suggesting the importance of endosomal escape. Our findings show that the transfection efficiency of DOTAP liposomes is mainly influenced by lipid composition and cell type, and not by size or zeta potential. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Conditions for gene transfection into the HL-60 human leukaemia cell line by electroporation

    Czech Academy of Sciences Publication Activity Database

    Pacherník, Jiří; Janík, Robert; Hofmanová, Jiřina; Bryja, Vítězslav; Kozubík, Alois

    2002-01-01

    Roč. 48, č. 4 (2002), s. 154-156 ISSN 0015-5500 R&D Projects: GA ČR GA524/99/0694; GA AV ČR IBS5004009; GA AV ČR KSK5011112 Institutional research plan: CEZ:AV0Z5004920 Keywords : leukaemia cell line HL-60 * electroporation * gene transfection Subject RIV: BO - Biophysics Impact factor: 0.615, year: 2002

  11. Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.

    Science.gov (United States)

    Schomaker, Markus; Heinemann, Dag; Kalies, Stefan; Willenbrock, Saskia; Wagner, Siegfried; Nolte, Ingo; Ripken, Tammo; Murua Escobar, Hugo; Meyer, Heiko; Heisterkamp, Alexander

    2015-02-03

    In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

  12. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes

    Directory of Open Access Journals (Sweden)

    Pappalardo JS

    2014-02-01

    Full Text Available Juan Sebastián Pappalardo,1–3 Cecilia A Langellotti,2 Sebastián Di Giacomo,1 Valeria Olivera,1 Valeria Quattrocchi,2 Patricia I Zamorano,1,2 William C Hartner,3 Tatyana S Levchenko,3 Vladimir P Torchilin3 1Virology Institute, Center for Research in Veterinary and Agronomic Sciences, National Institute for Agricultural Technology (INTA, Hurlingham, BA, Argentina; 2National Council for Scientific and Technical Research (CONICET, Autonomous City of Buenos Aires, Argentina; 3Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USA Abstract: Dendritic cells (DC are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp, is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD. The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6. Keywords: dendritic cell transfection, green fluorescent protein, bovine herpes virus 1 glycoprotein D, liposomes, TAT peptide, interleukin 6

  13. Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators.

    Science.gov (United States)

    Zhong, Cui-Li; Li, Guo-Ling; Mo, Jian-Xin; Quan, Rong; Wang, Hao-Qiang; Li, Zi-Cong; Wu, Zhen-Fang; Zhang, Xian-Wei

    2017-10-20

    To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM? 830 have the highest transfection efficiency when transfecting 10 μg supercoiled plasmids into PFFs, and 8 μg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.

  14. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation.

    Science.gov (United States)

    Jensen, Kirsty; Anderson, Jennifer A; Glass, Elizabeth J

    2014-04-15

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Optimization of transfection parameters for ultrasound/SonoVue microbubble-mediated hAng-1 gene delivery in vitro.

    Science.gov (United States)

    Zhou, Qing; Chen, Jin-Ling; Chen, Qian; Wang, Xiao; Deng, Qing; Hu, Bo; Guo, Rui-Qiang

    2012-12-01

    This study aimed to explore the effects of microbubble concentration, gene dosage, cell-microbubble mixing mode and fetal bovine serum (FBS) on gene delivery. 293T cells were transfected with Sonovue microbubbles carrying the hAng-1 gene via ultrasound irradiation. Various ultrasound exposure parameters and microbubble and DNA concentrations were investigated. In addition, FBS and the cell suspension or adherent mode was explored. Transfection efficiency and cell viability were used to determine the optimal transfection parameters. hAng-1 gene transfection efficiency gradually increased with elongation of ultrasound exposure and increasing microbubble concentration. However, if ultrasound irradiation exceeded 1.5 W/cm² and 30 sec or the microbubble concentration was over 20%, hAng-1 gene expression was significantly decreased, coupled with extensive cell death. Gene transfection levels were low under DNA concentrations less than 15 µg/ml. Furthermore, the gene transfer rate was significantly increased under cell suspension mode; FBS had no effect on hAng-1 gene transfection. The integrity of hAng-1 DNA was not affected by ultrasonic irradiation under optimal conditions. The optimal transfection parameters for the hAng-1 gene and Sonovue microbubble were ultrasound exposure of 1.5 W/cm² and 30 sec, 20% microbubbles, 15 µg/ml of DNA and under cell suspension mode.

  16. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    Directory of Open Access Journals (Sweden)

    Zhen Jin

    Full Text Available The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane. We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  17. Reduced repair of non-dimer photoproducts in a gene transfected into xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Protic-Sabljic, Miroslava; Kraemer, K.H.

    1986-01-01

    Cells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts was studied. It was found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m -2 of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA. (author)

  18. X-ray sensitive strains of CHO cells show decreased frequency of stable transfection

    International Nuclear Information System (INIS)

    Jeggo, P.; Smith, J.

    1987-01-01

    Six X-ray sensitive (xrs) strains of the Chinese hamster ovary cell line have previously been isolated and shown to have a defect in double strand break rejoining. In this study, these strains have been investigated for their ability to take up and integrate foreign DNA. All the xrs strains investigated so far have shown a decreased frequency of stable transfectants compared to their parent line, in experiments using the plasmid pSV2gpt, which contains the selectable bacterial gene, guanine phosphoribosyl transferase. This decreased frequency is observed over a wide range of DNA concentrations (0.1 to 20 μg DNA) but is more pronounced at higher DNA concentrations. In contrast, these xrs strains show the same level of transfection proficiency as the wild type parent using a transient transfection system with a plasmid containing the bacterial CAT (chloramphenicol acetyl transferase) gene. Since the level of CAT activity does not depend on integration of foreign DNA, this suggests that the xrs strains are able to take up the same amount of DNA as the parent strains, but have a defect in the integration of foreign DNA. Since this integration of foreign DNA probably occurs by non-homologous recombination, this may indicate a role of the xrs gene product in this process

  19. Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo.

    Science.gov (United States)

    Qiu, L; Zhang, L; Wang, L; Jiang, Y; Luo, Y; Peng, Y; Lin, L

    2012-07-01

    The study was to investigate the probability of increasing the transfection of the gene in tendons by ultrasound-targeted microbubble destruction (UTMD), and to search for the most suitable transfection conditions. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids was injected into rabbit Achilles tendons by different administration routes and the tendons were ultrasound pulse by different ultrasonic conditions in order to determine the most appropriate conditions. Then, the rabbits were divided into four groups: (1) ultrasound + microbubbles + plasmid; (2) ultrasound+ plasmid; (3) microbubble + plasmid; (4) plasmid only. EGFP expression in the tendons and other tissues, and the damage to tendon and paratenon were all observed. The results showed that EGFP expression in the tendon was higher by ultrasound pulse with 2 W cm(-2) of output intensity and a 20% duty cycle for 10 min. Local injection was determined to be the better administration route. Among the four groups, EGFP expression in Group 1 was higher than that in other groups. EGFP expression was highest on seventh day, then it gradually decrease over time, and lasted more than 56 days. EGFP expression was not found in other tissues. There was no obvious injury caused by UTMD. Under suitable conditions, it is feasible to use UTMD as a safe and effective gene transfection therapy for tendon injuries.

  20. Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

    Science.gov (United States)

    Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

    2011-01-01

    Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

  1. Structural mediation on polycation nanoparticles by sulfadiazine to enhance DNA transfection efficiency and reduce toxicity.

    Science.gov (United States)

    Long, Xingwen; Zhang, Zhihui; Han, Shangcong; Tang, Minjie; Zhou, Junhui; Zhang, Jianhua; Xue, Zhenyi; Li, Yan; Zhang, Rongxin; Deng, Liandong; Dong, Anjie

    2015-04-15

    Reducing the toxicity while maintaining high transfection efficiency is an important issue for cationic polymers as gene carriers in clinical application. In this paper, a new zwitterionic copolymer, polycaprolactone-g-poly(dimethylaminoethyl methyacrylate-co-sulfadiazine methacrylate) (PC-SDZ) with unique pH-sensitivity, was designed and prepared. The incorporation of sulfadiazine into poly(dimethylaminoethyl methacrylate) (PDMAEMA) chains successfully mediates the surface properties including compacter shell structure, lower density of positive charges, stronger proton buffer capability, and enhanced hydrophobicity, which lead to reduction in toxicity and enhancements in stability, cellular uptake, endosome escape, and transfection efficiency for the PC-SDZ2 nanoparticles (NPs)/DNA complexes. Excellent transfection efficiency at the optimal N/P ratio of 10 was observed for PC-SDZ2 NPs/DNA complexes, which was higher than that of the commercial reagent-branched polyethylenimine (PEI). The cytotoxicity was evaluated by CCK8 measurement, and the results showed significant reduction in cytotoxicity even at high concentration of complexes after sulfadiazine modification. Therefore, this work may demonstrate a new way of structural mediation of cationic polymer carriers for gene delivery with high efficiency and low toxicity.

  2. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

    Science.gov (United States)

    Pappalardo, Juan Sebastián; Langellotti, Cecilia A; Di Giacomo, Sebastián; Olivera, Valeria; Quattrocchi, Valeria; Zamorano, Patricia I; Hartner, William C; Levchenko, Tatyana S; Torchilin, Vladimir P

    2014-01-01

    Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

  3. Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

    Science.gov (United States)

    Talento, A; Nguyen, M; Law, S; Wu, J K; Poe, M; Blake, J T; Patel, M; Wu, T J; Manyak, C L; Silberklang, M

    1992-12-15

    Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

  4. Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.

    Science.gov (United States)

    Kuroda, Hitoshi; Kutner, Robert H; Bazan, Nicolas G; Reiser, Jakob

    2009-05-01

    During the past 12 years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.

  5. Drug Delivery and Cell Transfection Using Shock Waves Produced by Nanothermites

    Science.gov (United States)

    Gangopadhyay, Shubhra

    2009-06-01

    Shock waves have non-destructive life science applications in cell transfection and drug delivery. Based on molecular dynamics simulations, the shockwave causes transient compression of the cell membrane, which causes the hydrophobic interior of the lipid bilayer to become thinner. This allows diffusion of water molecules across the membrane. Recently, the nanothermite composition consisting of CuO nanorods and Al nanoparticles was shown to propagate at velocities in the same range as metallic azides and fulminates; however, the CuO/Al mixture produces lower pressure levels. An in vitro testing system was developed to deliver shock waves produced by nanothermites into cell suspensions and/or tissues. The plasmid encoded for production of green-fluorescent protein was delivered into cells including, among other types, chicken cardiomyocytes, cell lines (T47-D and Ins-1), and Arabidopsis plant cells. It was found that the nanothermite pressure impulses induced transfection resulting in production of green fluorescent protein in 99% of the cardiomyocytes. Additionally, transfected cell survival was evaluated, and the pressure impulses did not produce any elevated levels of cell death compared with control cell suspensions.

  6. Effects of Microbubble Size on Ultrasound-Mediated Gene Transfection in Auditory Cells

    Directory of Open Access Journals (Sweden)

    Ai-Ho Liao

    2014-01-01

    Full Text Available Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-facilitated ultrasound (US technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

  7. The Effect of Linear PEI on Characteristics and Transfection Efficiency of PEI-Based Cationic Nanoliposomes

    Directory of Open Access Journals (Sweden)

    Mohammad Ramezani

    2011-01-01

    Full Text Available Objective(sThe development of efficient and safe carrier system to transfer DNA into cells is essential in non-viral gene therapy. The aim of the present study was to evaluate the effect of linear polyetheneimine (lPEI (2500 Da on the physicochemical and biological properties of lipopolyplexes constructed from liposomes and lPEI. Materials and MethodsDifferent lipopolymers were synthesized from lPEI and acrylate derivatives. Nanocarriers were composed of the lipids (DOPE, DPPE and DOTAP and the synthesized lipopolymers. After characterization of the prepared vectors by determination of size and zeta potential, transfection activity was tested in Neuro2A cells. Ethidium bromide and MTT test were used to evaluate the DNA condensation ability and cytotoxicity of vectors, respectively. Results Vector’s size ranged from 95 to 337 nm and they had positive charge. The differences in DNA binding properties of lipopolyplexes were not significant. Among lipids, DOTAP showed better impact on transfection efficiency. The highest transfection activity was achieved by liposomal formulation consist of DOTAP and lipopolymer composed of lPEI and hexyl acrylate. The lipopolyplexes showed minimum cytotoxicity to the cultured cells in vitro. Conclusion The results of study confirmed that it is possible to improve gene expression using lipopolyplexes.

  8. Regulation of translocated c-myc genes transfected into plasmacytoma cells

    International Nuclear Information System (INIS)

    Feo, S.; Harvey, R.; Showe, L.; Croce, C.M.

    1986-01-01

    The authors have transfected two translocated c-myc oncogene clones, derived from two human lymphomas carrying the t(8;14) chromosome translocation, into mouse plasmacytoma cells to study the regulation of their expression. In one case, the transfected clone contained the two coding exons of the c-myc oncogene translocated to an immunoglobulin heavy-chain switch region; in the other case, the two coding exons were translocated 5' of the enhancer element located between the heavy-chain joining region (J/sub H/) and the switch region S/sub μ/. Nuclease S1 protection experiments indicate that only the c-myc translocated 5' of the enhancer element is transcribed in the plasmacytoma cells. Thus, 5'-truncation of the c-myc gene per se does not lead to c-myc deregulation. Further, since the level of c-myc transcripts in the parental human lymphoma cells was 3- to 4-fold higher than in the transfectants, it seems likely that additional elements within the heavy-chain locus may play a role in the enhancement of c-myc gene transcription in lymphoma cells

  9. Poly(ester-anhydride):poly(beta-amino ester) micro- and nanospheres: DNA encapsulation and cellular transfection.

    Science.gov (United States)

    Pfeifer, Blaine A; Burdick, Jason A; Little, Steve R; Langer, Robert

    2005-11-04

    Poly(ester-anhydride) delivery devices allow flexibility regarding carrier dimensions (micro- versus nanospheres), degradation rate (anhydride versus ester hydrolysis), and surface labeling (through the anhydride functional unit), and were therefore tested for DNA encapsulation and transfection of a macrophage P388D1 cell line. Poly(l-lactic acid-co-sebacic anhydride) and poly(l-lactic acid-co-adipic anhydride) were synthesized through melt condensation, mixed with 25 wt.% poly(beta-amino ester), and formulated with plasmid DNA (encoding firefly luciferase) into micro- and nanospheres using a double emulsion/solvent evaporation technique. The micro- and nanospheres were then characterized (size, morphology, zeta potential, DNA release) and assayed for DNA encapsulation and cellular transfection over a range of poly(ester-anhydride) copolymer ratios. Poly(ester-anhydride):poly(beta-amino ester) composite microspheres (6-12 microm) and nanospheres (449-1031 nm), generated with copolymers containing between 0 and 25% total polyanhydride content, encapsulated plasmid DNA (>or=20% encapsulation efficiency). Within this polyanhydride range, poly(adipic anhydride) copolymers provided DNA encapsulation at an increased anhydride content (10%, microspheres; 10-25%, nanospheres) compared to poly(sebacic anhydride) copolymers (1%, microspheres and nanospheres) with cellular transfection correlating with the observed DNA encapsulation.

  10. Inhibition of human colorectal adenocarcinoma cells with AdCMV-p53 gene transfection induced by irradiation

    International Nuclear Information System (INIS)

    Liu Bing; Min Fengling; Xie Yi; Zhou Qingming; Duan Xin; Chinese Academy of Sciences, Beijing; Zhang Hong; Li Wenjian; Hao Jifang; Zhou Guangming; Gao Qingxiang

    2006-01-01

    The effect of AdCMV-p53 gene transfection induced by γ-ray irradiation on human colorectal adenocarcinoma cells was investigated. The HT-29 cells were irradiated by 0.5, 1.0, 2.0 Gy 60 Co γ-rays, then were transfected with AdCMV-GFP (a replication of deficient recombinant adenoviral vector containing a CMV promoter and green fluorescent protein) or AdCMV-p53 (a replication of deficient recombinant adenoviral vector containing a CMV promoter and carrying human wild p53 gene). Cytotoxity was measured by clonogenic survival assay; apoptosis and the p53 expression were determined by flow cytometry. The results show that the pre-exposure of 0.5 Gy 60 Co γ-rays significantly enhanced the inhibition of HT-29 cells with AdCMV-53 transfection and promoted cell apoptosis. The inhibition rates for the groups of pre-exposure with 0.5 Gy and transfection with 40 and 80 MOI AdCMV-p53 were 50% and 20% higher than those for the groups of the mere transfection, and 40% more than the mere irradiation group. In the case of higher than 0.5 Gy pre-exposure, no significant difference was found between the pre-exposure with transfection group and the mere irradiation group. So 0.5 Gy pre-irradiation and AdCMV-p53 transfection obviously increases the inhibition of HT-29 cells with AdCMV-p53 transfection. The optimum condition is the lower than 1.0 Gy pre-exposure combined with the lower than 80 MOI AdCMV-p53 transfection. (authors)

  11. High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

    DEFF Research Database (Denmark)

    Adler, Andrew F; Speidel, Alessondra T; Christoforou, Nicolas

    2011-01-01

    Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter...... of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM...... and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems....

  12. Mammalian niche conservation through deep time.

    Directory of Open Access Journals (Sweden)

    Larisa R G DeSantis

    Full Text Available Climate change alters species distributions, causing plants and animals to move north or to higher elevations with current warming. Bioclimatic models predict species distributions based on extant realized niches and assume niche conservation. Here, we evaluate if proxies for niches (i.e., range areas are conserved at the family level through deep time, from the Eocene to the Pleistocene. We analyze the occurrence of all mammalian families in the continental USA, calculating range area, percent range area occupied, range area rank, and range polygon centroids during each epoch. Percent range area occupied significantly increases from the Oligocene to the Miocene and again from the Pliocene to the Pleistocene; however, mammalian families maintain statistical concordance between rank orders across time. Families with greater taxonomic diversity occupy a greater percent of available range area during each epoch and net changes in taxonomic diversity are significantly positively related to changes in percent range area occupied from the Eocene to the Pleistocene. Furthermore, gains and losses in generic and species diversity are remarkably consistent with ~2.3 species gained per generic increase. Centroids demonstrate southeastern shifts from the Eocene through the Pleistocene that may correspond to major environmental events and/or climate changes during the Cenozoic. These results demonstrate range conservation at the family level and support the idea that niche conservation at higher taxonomic levels operates over deep time and may be controlled by life history traits. Furthermore, families containing megafauna and/or terminal Pleistocene extinction victims do not incur significantly greater declines in range area rank than families containing only smaller taxa and/or only survivors, from the Pliocene to Pleistocene. Collectively, these data evince the resilience of families to climate and/or environmental change in deep time, the absence of

  13. Some principles of regeneration in mammalian systems.

    Science.gov (United States)

    Carlson, Bruce M

    2005-11-01

    This article presents some general principles underlying regenerative phenomena in vertebrates, starting with the epimorphic regeneration of the amphibian limb and continuing with tissue and organ regeneration in mammals. Epimorphic regeneration following limb amputation involves wound healing, followed shortly by a phase of dedifferentiation that leads to the formation of a regeneration blastema. Up to the point of blastema formation, dedifferentiation is guided by unique regenerative pathways, but the overall developmental controls underlying limb formation from the blastema generally recapitulate those of embryonic limb development. Damaged mammalian tissues do not form a blastema. At the cellular level, differentiation follows a pattern close to that seen in the embryo, but at the level of the tissue and organ, regeneration is strongly influenced by conditions inherent in the local environment. In some mammalian systems, such as the liver, parenchymal cells contribute progeny to the regenerate. In others, e.g., skeletal muscle and bone, tissue-specific progenitor cells constitute the main source of regenerating cells. The substrate on which regeneration occurs plays a very important role in determining the course of regeneration. Epimorphic regeneration usually produces an exact replica of the structure that was lost, but in mammalian tissue regeneration the form of the regenerate is largely determined by the mechanical environment acting on the regenerating tissue, and it is normally an imperfect replica of the original. In organ hypertophy, such as that occurring after hepatic resection, the remaining liver mass enlarges, but there is no attempt to restore the original form. (c) 2005 Wiley-Liss, Inc.

  14. Mammalian Synthetic Biology: Time for Big MACs.

    Science.gov (United States)

    Martella, Andrea; Pollard, Steven M; Dai, Junbiao; Cai, Yizhi

    2016-10-21

    The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-potentially up to chromosome scale-and the effective delivery of these large DNA payloads to the host cell. Random integration of large transgenes, encoding therapeutic proteins or genetic circuits into host chromosomes, has several drawbacks such as risks of insertional mutagenesis, lack of control over transgene copy-number and position-specific effects; these can compromise the intended functioning of genetic circuits. The development of a system orthogonal to the endogenous genome is therefore beneficial. Mammalian artificial chromosomes (MACs) are functional, add-on chromosomal elements, which behave as normal chromosomes-being replicating and portioned to daughter cells at each cell division. They are deployed as useful gene expression vectors as they remain independent from the host genome. MACs are maintained as a single-copy and can accommodate multiple gene expression cassettes of, in theory, unlimited DNA size (MACs up to 10 megabases have been constructed). MACs therefore enabled control over ectopic gene expression and represent an excellent platform to rapidly prototype and characterize novel synthetic gene circuits without recourse to engineering the host genome. This review describes the obstacles synthetic biologists face when working with mammalian systems and how the development of improved MACs can overcome these-particularly given the spectacular advances in DNA synthesis and assembly that are fuelling this research area.

  15. Preservation of mammalian germ plasm by freezing

    Energy Technology Data Exchange (ETDEWEB)

    Mazur, P.

    1978-01-01

    Embryos of several mammalian species can be frozen to -196/sup 0/C (or below) by procedures that result in the thawed embryos being indistinguishable from their unfrozen counterparts. The survival often exceeds 90%, and in liquid nitrogen it should remain at that high level for centuries. Sublethal biochemical changes are also precluded at -196/sup 0/C. No developmental abnormalities have been detected in mouse offspring derived from frozen-thawed embryos, and, since all the manipulations are carried out on the preimplantation stages, none would be expected.

  16. Modeling Exposure of Mammalian Predatorsto Anticoagulant Rodenticides

    DEFF Research Database (Denmark)

    Topping, Christopher John; Elmeros, Morten

    2016-01-01

    and creates an exposure map based on spatio-temporal modelling of movement of mice-vectored AR (based on Apodemus flavicollis). Simulated predator territories are super-imposed over this exposure map to create an exposure index. Predictions from the model concur with field studies of AR prevalence both before...... high. We postulate that this is caused by widespread exposure due to widespread use of AR in Denmark in and around buildings. To investigate this theory a spatio-temporal model of AR use and mammalian predator distribution was created. This model was supported by data from an experimental study of mice...

  17. Mammalian developmental genetics in the twentieth century.

    Science.gov (United States)

    Artzt, Karen

    2012-12-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas.

  18. Nutrient acquisition strategies of mammalian cells.

    Science.gov (United States)

    Palm, Wilhelm; Thompson, Craig B

    2017-06-07

    Mammalian cells are surrounded by diverse nutrients, such as glucose, amino acids, various macromolecules and micronutrients, which they can import through transmembrane transporters and endolysosomal pathways. By using different nutrient sources, cells gain metabolic flexibility to survive periods of starvation. Quiescent cells take up sufficient nutrients to sustain homeostasis. However, proliferating cells depend on growth-factor-induced increases in nutrient uptake to support biomass formation. Here, we review cellular nutrient acquisition strategies and their regulation by growth factors and cell-intrinsic nutrient sensors. We also discuss how oncogenes and tumour suppressors promote nutrient uptake and thereby support the survival and growth of cancer cells.

  19. Better Smelling Through Genetics: Mammalian Odor Perception

    OpenAIRE

    Keller, Andreas; Vosshall, Leslie B.

    2008-01-01

    The increasing availability of genomic and genetic tools to study olfaction—the sense of smell—has brought important new insights into how this chemosensory modality functions in different species. Newly sequenced mammalian genomes—from platypus to dog—have made it possible to infer how smell has evolved to suit the needs of a given species and how variation within a species may affect individual olfactory perception. This review will focus on recent advances in the genetics and genomics of m...

  20. microRNA-mediated messenger RNA deadenylation contributes to translational repression in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Traude H Beilharz

    2009-08-01

    Full Text Available Animal microRNAs (miRNAs typically regulate gene expression by binding to partially complementary target sites in the 3' untranslated region (UTR of messenger RNA (mRNA reducing its translation and stability. They also commonly induce shortening of the mRNA 3' poly(A tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3' UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.

  1. Ecology and evolution of mammalian biodiversity.

    Science.gov (United States)

    Jones, Kate E; Safi, Kamran

    2011-09-12

    Mammals have incredible biological diversity, showing extreme flexibility in eco-morphology, physiology, life history and behaviour across their evolutionary history. Undoubtedly, mammals play an important role in ecosystems by providing essential services such as regulating insect populations, seed dispersal and pollination and act as indicators of general ecosystem health. However, the macroecological and macroevolutionary processes underpinning past and present biodiversity patterns are only beginning to be explored on a global scale. It is also particularly important, in the face of the global extinction crisis, to understand these processes in order to be able to use this knowledge to prevent future biodiversity loss and loss of ecosystem services. Unfortunately, efforts to understand mammalian biodiversity have been hampered by a lack of data. New data compilations on current species' distributions, ecologies and evolutionary histories now allow an integrated approach to understand this biodiversity. We review and synthesize these new studies, exploring the past and present ecology and evolution of mammalian biodiversity, and use these findings to speculate about the mammals of our future.

  2. Redox regulation of mammalian sperm capacitation

    Directory of Open Access Journals (Sweden)

    Cristian O′Flaherty

    2015-01-01

    Full Text Available Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P H for sperm capacitation. Peroxiredoxins (PRDXs are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility.

  3. Redox regulation of mammalian sperm capacitation

    Science.gov (United States)

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  4. Structure and function in mammalian societies.

    Science.gov (United States)

    Clutton-Brock, Tim

    2009-11-12

    Traditional interpretations of the evolution of animal societies have suggested that their structure is a consequence of attempts by individuals to maximize their inclusive fitness within constraints imposed by their social and physical environments. In contrast, some recent re-interpretations have argued that many aspects of social organization should be interpreted as group-level adaptations maintained by selection operating between groups or populations. Here, I review our current understanding of the evolution of mammalian societies, focusing, in particular, on the evolution of reproductive strategies in societies where one dominant female monopolizes reproduction in each group and her offspring are reared by other group members. Recent studies of the life histories of females in these species show that dispersing females often have little chance of establishing new breeding groups and so are likely to maximize their inclusive fitness by helping related dominants to rear their offspring. As in eusocial insects, increasing group size can lead to a progressive divergence in the selection pressures operating on breeders and helpers and to increasing specialization in their behaviour and life histories. As yet, there is little need to invoke group-level adaptations in order to account for the behaviour of individuals or the structure of mammalian groups.

  5. Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

    Science.gov (United States)

    Osorio, Johan S; Bionaz, Massimo

    2017-08-30

    Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor

  6. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection

    Science.gov (United States)

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-01-01

    Abstract. In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage. PMID:25069006

  7. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection.

    Science.gov (United States)

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-01-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  8. Synthetic RNA Controllers for Programming Mammalian Cell Fate and Function

    Science.gov (United States)

    2015-11-04

    Final report for “Synthetic RNA controllers for programming mammalian cell fate and function” Principal Investigator: Christina D. Smolke...SUBTITLE Synthetic RNA controllers for programming mammalian cell fate and function 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18   2 Synthetic RNA controllers for programming mammalian cell fate and function Task 1

  9. INFLUENCE OF TEMPERATURE ON AN ESTROGEN-RESPONSIVE RAINBOW TROUT CELL TRANSFECTION ASSAY

    Science.gov (United States)

    One uncertainty in extrapolating estrogenic effects in mammalian systems to those in fish and wildlife is the influence that temperature has on these effects. A reporter gene assay in cultured rainbow trout cell lines was used to determine the influence of temperature on the exp...

  10. Acceleration of gene transfection efficiency in neuroblastoma cells through polyethyleneimine/poly(methyl methacrylate) core-shell magnetic nanoparticles

    Science.gov (United States)

    Tencomnao, Tewin; Klangthong, Kewalin; Pimpha, Nuttaporn; Chaleawlert-umpon, Saowaluk; Saesoo, Somsak; Woramongkolchai, Noppawan; Saengkrit, Nattika

    2012-01-01

    Background The purpose of this study was to demonstrate the potential of magnetic poly(methyl methacrylate) (PMMA) core/polyethyleneimine (PEI) shell (mag-PEI) nanoparticles, which possess high saturation magnetization for gene delivery. By using mag-PEI nanoparticles as a gene carrier, this study focused on evaluation of transfection efficiency under magnetic induction. The potential role of this newly synthesized nanosphere for therapeutic delivery of the tryptophan hydroxylase-2 (TPH-2) gene was also investigated in cultured neuronal LAN-5 cells. Methods The mag-PEI nanoparticles were prepared by one-step emulsifier-free emulsion polymerization, generating highly loaded and monodispersed magnetic polymeric nanoparticles bearing an amine group. The physicochemical properties of the mag-PEI nanoparticles and DNA-bound mag-PEI nanoparticles were investigated using the gel retardation assay, atomic force microscopy, and zeta size measurements. The gene transfection efficiencies of mag-PEI nanoparticles were evaluated at different transfection times. Confocal laser scanning microscopy confirmed intracellular uptake of the magnetoplex. The optimal conditions for transfection of TPH-2 were selected for therapeutic gene transfection. We isolated the TPH-2 gene from the total RNA of the human medulla oblongata and cloned it into an expression vector. The plasmid containing TPH-2 was subsequently bound onto the surfaces of the mag-PEI nanoparticles via electrostatic interaction. Finally, the mag-PEI nanoparticle magnetoplex was delivered into LAN-5 cells. Reverse-transcriptase polymerase chain reaction was performed to evaluate TPH-2 expression in a quantitative manner. Results The study demonstrated the role of newly synthesized high-magnetization mag-PEI nanoparticles for gene transfection in vitro. The expression signals of a model gene, luciferase, and a therapeutic gene, TPH-2, were enhanced under magnetic-assisted transfection. An in vitro study in neuronal cells

  11. Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

    Directory of Open Access Journals (Sweden)

    Jing Dan

    2017-03-01

    Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

  12. High‐throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies

    Science.gov (United States)

    Nguyen, Albert; Beyersdorf, Jared; Riethoven, Jean‐Jack

    2016-01-01

    Abstract Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated

  13. Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

    Science.gov (United States)

    Uchida, Hirokuni; Itaka, Keiji; Nomoto, Takahiro; Ishii, Takehiko; Suma, Tomoya; Ikegami, Masaru; Miyata, Kanjiro; Oba, Makoto; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2014-09-03

    Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

  14. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    Science.gov (United States)

    Yang, Xuechao; Walboomers, X Frank; van den Dolder, Juliette; Yang, Fang; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.

  15. Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

    OpenAIRE

    Dong, Yuzhen; Yang, Libin; Yang, Lin; Zhao, Hongxing; Zhang, Chao; Wu, Dapeng

    2014-01-01

    Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesenchymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal ...

  16. The Adaptive Response to Intestinal Oxidative Stress in Mammalian Hibernation

    National Research Council Canada - National Science Library

    Carey, Hannah V

    2006-01-01

    The goal of this project is demonstrate how mammalian hibernators utilize the physiologic consequences of metabolic depression, which include changes in mitochondrial function, low body temperatures (Tb...

  17. Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1.

    Science.gov (United States)

    Nukuzuma, Souichi; Nakamichi, Kazuo; Kameoka, Masanori; Sugiura, Shigeki; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2010-12-01

    The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV. © 2010 The Societies and Blackwell Publishing Asia Pty Ltd.

  18. Water-resistant, monodispersed and stably luminescent CsPbBr3/CsPb2Br5 core-shell-like structure lead halide perovskite nanocrystals

    Science.gov (United States)

    Qiao, Bo; Song, Pengjie; Cao, Jingyue; Zhao, Suling; Shen, Zhaohui; Gao, Di; Liang, Zhiqin; Xu, Zheng; Song, Dandan; Xu, Xurong

    2017-11-01

    Lead halide perovskite materials are thriving in optoelectronic applications due to their excellent properties, while their instability due to the fact that they are easily hydrolyzed is still a bottleneck for their potential application. In this work, water-resistant, monodispersed and stably luminescent cesium lead bromine perovskite nanocrystals coated with CsPb2Br5 were obtained using a modified non-stoichiometric solution-phase method. CsPb2Br5 2D layers were coated on the surface of CsPbBr3 nanocrystals and formed a core-shell-like structure in the synthetic processes. The stability of the luminescence of the CsPbBr3 nanocrystals in water and ethanol atmosphere was greatly enhanced by the photoluminescence-inactive CsPb2Br5 coating with a wide bandgap. The water-stable enhanced nanocrystals are suitable for long-term stable optoelectronic applications in the atmosphere.

  19. Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

    Science.gov (United States)

    Yoon, Sangpil; Wang, Yingxiao; Shung, K. K.

    2016-03-01

    Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.

  20. Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease.

    Science.gov (United States)

    Ye, Lei; Haider, Husnain Kh; Esa, Wahidah Bte; Su, Liping; Law, Peter K; Zhang, Wei; Lim, Yeanteng; Poh, Kian Keong; Sim, Eugene K W

    2010-01-01

    The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

  1. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    International Nuclear Information System (INIS)

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong; Wan, Min; Li, Yong-Qiang; Zhang, Rong-Ying; Zhao, Yuan-Di

    2012-01-01

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  2. In vivo molecular imaging and radionuclide (131I therapy of human nasopharyngeal carcinoma cells transfected with a lentivirus expressing sodium iodide symporter.

    Directory of Open Access Journals (Sweden)

    Shuo Shi

    Full Text Available Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC, novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using 125I and 131I therapy in vivo.We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP under the control of the human elongation factor-1α (EF1α promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; 125I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with 131I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more 125I than CNE-2Z cells and xenografts. In vitro, 131I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, 131I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of 131I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.

  3. Abrogation of radiation-inducible telomerase upregulation in HPV16 E6 transfectants of human lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Neuhof, D.; Auberger, F.; Ruess, A.; Weber, K.J. [Radiobiology Research Lab., Dept. of Clinical Radiology, Univ. of Heidelberg (Germany); Wenz, F. [Section for Radiation Oncology, Univ. Clinic Mannheim (Germany)

    2004-01-01

    Background: telomerase activity in a human lymphoblastoid cell line with wild-type p53 status (TK6) was previously shown to be rapidly induced by ionizing radiation doses as low as 10 cgy. Since this low-dose response was absent in a closely related cell line overexpressing a mutant form of p53 (WTK1), the putative involvement of p53 was further investigated using stable human papillomavirus 16 (HPV16) E6 transfectants of these cell lines. The E6 product mediates rapid degradation of wild-type p53, but has also been found to upregulate telomerase. Material and methods: telomerase activity in HPV16 E6 transfectants of the human lymphoblastoid cell lines TK6 and WTK1 was measured by PCR/ELISA and was quantified using internal standards (titration by cell number) run within each separate assay. Mean telomere length was determined by southern hybridization of terminal restriction fragments with a biotin-labeled telomeric DNA probe. Results: the TK6E6 and the WTK1E6 cells exhibited higher baseline telomerase activities than the parental cells. This was also accompanied by increased telomere lengths. Radiation exposure (up to 10 gy) was unable to significantly further enhance telomerase activities, although the dynamic range of the assay would have allowed to record higher signals. Conclusion: the lacking radiation induction of telomerase activities in the E6 transfectants could reflect saturation, if E6 and radiation would share a common pathway of telomerase upregulation. Present evidence from the literature, however, suggests that E6 mediates telomerase reverse transcriptase (TERT) subunit transcriptional activation, whereas radiation signals to posttranscriptional/posttranslational control of telomerase activity. Therefore, the present data enforce the previous hypothesis of a p53 dependence of telomerase upregulation by low doses of radiation and its abrogation, likely due to p53 degradation, in E6-expressing cells. (orig.)

  4. Cationic nanoparticles with quaternary ammonium-functionalized PLGA-PEG-based copolymers for potent gene transfection

    Science.gov (United States)

    Wang, Yan-Hsung; Fu, Yin-Chih; Chiu, Hui-Chi; Wang, Chau-Zen; Lo, Shao-Ping; Ho, Mei-Ling; Liu, Po-Len; Wang, Chih-Kuang

    2013-11-01

    The objective of the present work was to develop new cationic nanoparticles (cNPs) with amphiphilic cationic copolymers for the delivery of plasmid DNA (pDNA). Cationic copolymers were built on the synthesis of quaternary ammonium salt compounds from diethylenetriamine (DETA) to include the positively charged head group and amphiphilic multi-grafts. PLGA- phe-PEG- qDETA (PPD), phe-PEG- qDETA-PLGA (PDP), and PLGA- phe-PEG- qDETA-PLGA (PPDP) cationic copolymers were created by this moiety of DETA quaternary ammonium, heterobifunctional polyethylene glycol (COOH-PEG-NH2), phenylalanine ( phe), and poly(lactic- co-glycolic acid) (PLGA). These new cNPs were prepared by the water miscible solvent displacement method. They exhibit good pDNA binding ability, as shown in a retardation assay that occurred at a particle size of 217 nm. The zeta potential was approximately +21 mV when the cNP concentration was 25 mg/ml. The new cNPs also have a better buffering capacity than PLGA NPs. However, the pDNA binding ability was demonstrated starting at a weight ratio of approximately 6.25 cNPs/pDNA. Gene transfection results showed that these cNPs had transfection effects similar to those of Lipofectamine 2000 in 293T cells. Furthermore, cNPs can also transfect human adipose-derived stem cells. The results indicate that the newly developed cNP is a promising candidate for a novel gene delivery vehicle.

  5. Cationic nanoparticles with quaternary ammonium-functionalized PLGA–PEG-based copolymers for potent gene transfection

    International Nuclear Information System (INIS)

    Wang, Yan-Hsung; Fu, Yin-Chih; Chiu, Hui-Chi; Wang, Chau-Zen; Lo, Shao-Ping; Ho, Mei-Ling; Liu, Po-Len; Wang, Chih-Kuang

    2013-01-01

    The objective of the present work was to develop new cationic nanoparticles (cNPs) with amphiphilic cationic copolymers for the delivery of plasmid DNA (pDNA). Cationic copolymers were built on the synthesis of quaternary ammonium salt compounds from diethylenetriamine (DETA) to include the positively charged head group and amphiphilic multi-grafts. PLGA-phe-PEG-qDETA (PPD), phe-PEG-qDETA-PLGA (PDP), and PLGA-phe-PEG-qDETA-PLGA (PPDP) cationic copolymers were created by this moiety of DETA quaternary ammonium, heterobifunctional polyethylene glycol (COOH-PEG-NH 2 ), phenylalanine (phe), and poly(lactic-co-glycolic acid) (PLGA). These new cNPs were prepared by the water miscible solvent displacement method. They exhibit good pDNA binding ability, as shown in a retardation assay that occurred at a particle size of ∼217 nm. The zeta potential was approximately +21 mV when the cNP concentration was 25 mg/ml. The new cNPs also have a better buffering capacity than PLGA NPs. However, the pDNA binding ability was demonstrated starting at a weight ratio of approximately 6.25 cNPs/pDNA. Gene transfection results showed that these cNPs had transfection effects similar to those of Lipofectamine 2000 in 293T cells. Furthermore, cNPs can also transfect human adipose-derived stem cells. The results indicate that the newly developed cNP is a promising candidate for a novel gene delivery vehicle

  6. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z.; Wientjes, M. Guillaume; Cole, David J.; Au, Jessie L.-S.

    2014-01-01

    Cancers originating from digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors [1–3]. TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancer. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer. PMID:24462901

  7. Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors.

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Wang, Junfeng; Cui, Minjian; Yeung, Bertrand Z; Cole, David J; Wientjes, M Guillaume; Au, Jessie L-S

    2015-10-28

    The major barrier for using small interfering RNA (siRNA) as cancer therapeutics is the inadequate delivery and transfection in solid tumors. We have previously shown that paclitaxel tumor priming, by inducing apoptosis, expands the tumor interstitial space, improves the penetration and dispersion of nanoparticles and siRNA-lipoplexes in 3-dimensional tumor histocultures, and promotes the delivery and transfection efficiency of siRNA-lipoplexes under the locoregional setting in vivo (i.e., intraperitoneal treatment of intraperitoneal tumors). The current study evaluated whether tumor priming is functional for systemically delivered siRNA via intravenous injection, which would subject siRNA to several additional delivery barriers and elimination processes. We used the same pegylated cationic (PCat)-siRNA lipoplexes as in the intraperitoneal study to treat mice bearing subcutaneous human pancreatic Hs766T xenograft tumors. The target gene was survivin, an inducible chemoresistance gene. The results show single agent paclitaxel delayed tumor growth but also significantly induced the survivin protein level in residual tumors, whereas addition of PCat-siSurvivin completely reversed the paclitaxel-induced survivin and enhanced the paclitaxel activity (ppriming, by promoting the interstitial transport and cytoplasmic release, is critical to promote the delivery and transfection of siRNA in vivo. In addition, because paclitaxel has broad spectrum activity and is used to treat multiple types of solid tumors including the hard-to-treat pancreatic cancer, the synergistic paclitaxel+siSurvivin combination represents a potentially useful chemo-gene therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Protic-Sabljic, M.; Kraemer, K.H.

    1985-01-01

    The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D 0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA

  9. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    OpenAIRE

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using soma...

  10. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  11. Improving the Gene Transfection in Human Embryonic Stem Cells: Balancing with Cytotoxicity and Pluripotent Maintenance.

    Science.gov (United States)

    Luo, Chunhua; Lü, Dongyuan; Pan, Jun; Long, Mian

    2016-04-06

    Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however, the transduction efficiency remains very low. Although existing evidence revealed the type, size, and zeta potential of vector affect gene transfection efficiency in cells, the systematic study in hESCs is scarce. In this study, using poly(amidoamine) (PAMAM) dendrimers ended with amine, hydroxyl, or carboxyl as model, we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5, G7, G4.5COOH, and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential, respectively, whereas G1 was slightly negative, and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs, which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers, especially with higher generations, were detrimental to the growth and pluripotent maintenance of hESCs. In contrast, similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity, in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size, concentration, and zeta potential, particularly the identity and position of groups, as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary, which helps to better design an effective vector in hESC gene transduction.

  12. Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds

    Science.gov (United States)

    Petrakova, V.; Benson, V.; Buncek, M.; Fiserova, A.; Ledvina, M.; Stursa, J.; Cigler, P.; Nesladek, M.

    2016-06-01

    Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for

  13. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    Energy Technology Data Exchange (ETDEWEB)

    Wu Xuewen; Ding Dalian [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Jiang Haiyan [State University of New York at Buffalo, Center for Hearing and Deafness (United States); XingXiaowei [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Huang, Suping [Central South University, State Key Laboratory of Powder Metallurgy (China); Liu Hong [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Chen Zhedong [Central South University, State Key Laboratory of Powder Metallurgy (China); Sun Hong, E-mail: shjhaj@vip.163.com [Central South University, Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital (China)

    2012-01-15

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 {+-} 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of

  14. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    International Nuclear Information System (INIS)

    Wu Xuewen; Ding Dalian; Jiang Haiyan; XingXiaowei; Huang, Suping; Liu Hong; Chen Zhedong; Sun Hong

    2012-01-01

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for

  15. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.

    Directory of Open Access Journals (Sweden)

    Daiane P Oldiges

    2016-12-01

    Full Text Available The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST. The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase, and HlGST fused to the MSA-1 (merozoite surface antigen 1 signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on Hl

  16. Real-time impedimetric monitoring of Poly(ethylenimine)s-mediated cytotoxicity during gene transfection

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, Marco; Heiskanen, Arto

    Poly(ethylenimine)s (PEIs) are able to condense DNA and RNA into stable toroidal and globular nanostructures (polyplexes) and are among the most efficient and promising synthetic transfectants, but they induce severe cytotoxicity. The mechanisms of PEI-mediated cytotoxicity have not been fully...... delineated but PEI toxicity appears to predominantly depend on membrane perturbing effects in cellular compartments in which they accumulate. Electrochemical Impedance Spectroscopy (EIS) is used as a non-invasive biophysical approach for the investigation of the electrical properties of biological materials...

  17. Aminooxy analog of histamine is an efficient inhibitor of mammalian L-histidine decarboxylase: combined in silico and experimental evidence.

    Science.gov (United States)

    Castro-Oropeza, R; Pino-Ángeles, A; Khomutov, M A; Urdiales, J L; Moya-García, A A; Vepsäläinen, J; Persson, L; Sarabia, F; Khomutov, A; Sánchez-Jiménez, F

    2014-03-01

    Histamine plays highlighted roles in the development of many common, emergent and rare diseases. In mammals, histamine is formed by decarboxylation of L-histidine, which is catalyzed by pyridoxal-5'-phosphate (PLP) dependent histidine decarboxylase (HDC, EC 4.1.1.22). The limited availability and stability of the protein have delayed the characterization of its structure-function relationships. Our previous knowledge on mammalian HDC, derived from both in silico and experimental approaches, indicates that an effective competitive inhibitor should be capable to form an "external aldimine-like structure" and have an imidazole group, or its proper mimetic, which provides additional affinity of PLP-inhibitor adduct to the HDC active center. This is confirmed using HEK-293 cells transfected to express human HDC and the aminooxy analog of histidine, 4(5)-aminooxymethylimidazole (O-IMHA, IC₅₀ ≈ 2 × 10(-7) M) capable to form a PLP-inhibitor complex (oxime) in the enzyme active center. Taking advantage of the availability of the human HDC X-ray structure, we have also determined the potential interactions that could stabilize this oxime in the active site of mammalian HDC.

  18. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis.

    Science.gov (United States)

    Yagnik, Bhrugu; Padh, Harish; Desai, Priti

    2016-04-01

    Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line.

    Science.gov (United States)

    Yusufi, Faraaz Noor Khan; Lakshmanan, Meiyappan; Ho, Ying Swan; Loo, Bernard Liat Wen; Ariyaratne, Pramila; Yang, Yuansheng; Ng, Say Kong; Tan, Tessa Rui Min; Yeo, Hock Chuan; Lim, Hsueh Lee; Ng, Sze Wai; Hiu, Ai Ping; Chow, Chung Ping; Wan, Corrine; Chen, Shuwen; Teo, Gavin; Song, Gao; Chin, Ju Xin; Ruan, Xiaoan; Sung, Ken Wing Kin; Hu, Wei-Shou; Yap, Miranda Gek Sim; Bardor, Muriel; Nagarajan, Niranjan; Lee, Dong-Yup

    2017-05-24

    Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Neuronal Circuitry Mechanisms Regulating Adult Mammalian Neurogenesis

    Science.gov (United States)

    Song, Juan; Olsen, Reid H.J.; Sun, Jiaqi; Ming, Guo-li; Song, Hongjun

    2017-01-01

    The adult mammalian brain is a dynamic structure, capable of remodeling in response to various physiological and pathological stimuli. One dramatic example of brain plasticity is the birth and subsequent integration of newborn neurons into the existing circuitry. This process, termed adult neurogenesis, recapitulates neural developmental events in two specialized adult brain regions: the lateral ventricles of the forebrain. Recent studies have begun to delineate how the existing neuronal circuits influence the dynamic process of adult neurogenesis, from activation of quiescent neural stem cells (NSCs) to the integration and survival of newborn neurons. Here, we review recent progress toward understanding the circuit-based regulation of adult neurogenesis in the hippocampus and olfactory bulb. PMID:27143698

  1. X-rays sensitive mammalian cell mutant

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi

    1982-01-01

    A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

  2. Structures of mammalian cytosolic quinone reductases.

    Science.gov (United States)

    Foster, C E; Bianchet, M A; Talalay, P; Faig, M; Amzel, L M

    2000-08-01

    The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.

  3. Mechanosensor Channels in Mammalian Somatosensory Neurons

    Directory of Open Access Journals (Sweden)

    Patrick Delmas

    2007-09-01

    Full Text Available Mechanoreceptive sensory neurons innervating the skin, skeletal muscles andviscera signal both innocuous and noxious information necessary for proprioception, touchand pain. These neurons are responsible for the transduction of mechanical stimuli intoaction potentials that propagate to the central nervous system. The ability of these cells todetect mechanical stimuli impinging on them relies on the presence of mechanosensitivechannels that transduce the external mechanical forces into electrical and chemical signals.Although a great deal of information regarding the molecular and biophysical properties ofmechanosensitive channels in prokaryotes has been accumulated over the past two decades,less is known about the mechanosensitive channels necessary for proprioception and thesenses of touch and pain. This review summarizes the most pertinent data onmechanosensitive channels of mammalian somatosensory neurons, focusing on theirproperties, pharmacology and putative identity.

  4. Mammalian synthetic biology for studying the cell.

    Science.gov (United States)

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  5. Peromyscus as a Mammalian Epigenetic Model

    Directory of Open Access Journals (Sweden)

    Kimberly R. Shorter

    2012-01-01

    Full Text Available Deer mice (Peromyscus offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential.

  6. Engineered Trehalose Permeable to Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Alireza Abazari

    Full Text Available Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre and trehalose tetraacetate (4-O-Ac-Tre. Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

  7. The architecture of mammalian ribosomal protein promoters

    Directory of Open Access Journals (Sweden)

    Perry Robert P

    2005-02-01

    Full Text Available Abstract Background Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. Results A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y2C+1TY(T2(Y3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. Conclusions This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.

  8. Phototransfection of mammalian cells using femtosecond laser pulses: optimization and applicability to stem cell differentiation

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-07-01

    Full Text Available , the influence of the cell passage number on the transfection efficiency is explored and, second, the ability to enhance the transfection efficiency via whole culture treatments. Our results should encourage wider uptake of this methodology...

  9. Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

    Science.gov (United States)

    Drew, Liam J.; Fusi, Stefano; Hen, René

    2013-01-01

    In the adult mammalian brain, newly generated neurons are continuously incorporated into two networks: interneurons born in the subventricular zone migrate to the olfactory bulb, whereas the dentate gyrus (DG) of the hippocampus integrates locally born principal neurons. That the rest of the mammalian brain loses significant neurogenic capacity…

  10. Mammalian gastrointestinal parasites in rainforest remnants of the ...

    Indian Academy of Sciences (India)

    Parasite prevalence (%) of nonhuman mammalian species of the tenstudy sites in Anamalai Tiger. Reserve, Western Ghats, India. Supplementary table 2. Percent prevalence of parasite taxa in 17 mammalian hosts from fragmented rainforest landscape of. Anamalai tiger reserve, Western Ghats, India. Supplementary table ...

  11. Non - flying mammalian fauna of Ampijoroa, Ankarafantsika National ...

    African Journals Online (AJOL)

    Non - flying mammalian fauna of Ampijoroa, Ankarafantsika National Park. R Ito, F Rakotondraparany, H Sato. Abstract. There is no list of the mammalian fauna of Ampijoroa Forest Station, a dry deciduous forest within Ankarafantsika National Park. We set Sherman traps and pitfall traps and carried out transect surveys to ...

  12. Bioinformatic analyses of kappa casein gene in mammalian ...

    African Journals Online (AJOL)

    Kappa casein (CSN3) gene is a variant of the milk protein highly conserved in mammalian species. Genetic variations in CSN3 gene of six mammalian livestock species were investigated using bioinformatics approach. A total of twenty-seven CSN3 gene sequences with corresponding amino acids belonging to the six ...

  13. Mammalian gastrointestinal parasites in rainforest remnants of the ...

    Indian Academy of Sciences (India)

    Supplementary table 1. Parasite prevalence (%) of nonhuman mammalian species of the tenstudy sites in Anamalai Tiger. Reserve, Western Ghats, India. Supplementary table 2. Percent prevalence of parasite taxa in 17 mammalian hosts from fragmented rainforest landscape of. Anamalai tiger reserve, Western Ghats, ...

  14. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Science.gov (United States)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-05-01

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  15. Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles

    Science.gov (United States)

    Tenkumo, Taichi; Kamano, Yuya; Egusa, Hiroshi; Sasaki, Keiichi

    2017-01-01

    Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP), the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8), which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8)-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220–580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC) and human osteoblasts (hOB) in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB. PMID:29145481

  16. Protective Effects of Moringa oleifera on HBV Genotypes C and H Transiently Transfected Huh7 Cells

    Science.gov (United States)

    Feustel, Sina; Ayón-Pérez, Fabiola; Sandoval-Rodriguez, Ana; Rodríguez-Echevarría, Roberto; Contreras-Salinas, Homero

    2017-01-01

    Chronic hepatitis B infection treatment implicates a long-lasting treatment. M. oleifera extracts contain compounds with antiviral, antioxidant, and antifibrotic properties. In this study, the effect of M. oleifera was evaluated in Huh7 cells expressing either HBV genotypes C or H for the antiviral, antifibrotic, anti-inflammatory, and antioxidative responses. Huh7 cells were treated with an aqueous extract of M. oleifera (leaves) at doses of 0, 30, 45, or 60 μg/mL. The replicative virus and TGF-β1, CTGF, CAT, IFN-β1, and pgRNA expressions were measured by real time. HBsAg and IL-6 titers were determined by ELISA. CTGF, TGF-β1, IFN-β1, and pgRNA expressions decreased with M. oleifera treatment irrespective of the HBV genotype. HBsAg secretion in the supernatant of transfected Huh7 cells with both HBV genotypes was decreased regardless of the dose of M. oleifera. Similar effect was observed in proinflammatory cytokine IL-6, which had a tendency to decrease at 24 hours of treatment. Transfection with both HBV genotypes strongly decreased CAT expression, which is retrieved with M. oleifera treatment. M. oleifera treatment reduced fibrosis markers, IL-6, and HBsAg secretion in HBV genotypes C and H. However, at the level of replication, only HBV-DNA genotype C was slightly reduced with this treatment. PMID:29214184

  17. Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

    Science.gov (United States)

    Masani, Mat Yunus Abdul; Noll, Gundula A.; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

    2014-01-01

    Background Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. PMID:24821306

  18. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    Energy Technology Data Exchange (ETDEWEB)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad, E-mail: amtamadon@gmail.com [Shiraz University of Medical Sciences, Department of Pharmaceutics, School of Pharmacy (Iran, Islamic Republic of)

    2016-05-15

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and {sup 1}H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol{sub 40 %}) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  19. Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

    Science.gov (United States)

    Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

    2012-01-01

    The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

  20. Specific transfection of inflamed brain by macrophages: a new therapeutic strategy for neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Matthew J Haney

    Full Text Available The ability to precisely upregulate genes in inflamed brain holds great therapeutic promise. Here we report a novel class of vectors, genetically modified macrophages that carry reporter and therapeutic genes to neural cells. Systemic administration of macrophages transfected ex vivo with a plasmid DNA (pDNA encoding a potent antioxidant enzyme, catalase, produced month-long expression levels of catalase in the brain resulting in three-fold reductions in inflammation and complete neuroprotection in mouse models of Parkinson's disease (PD. This resulted in significant improvements in motor functions in PD mice. Mechanistic studies revealed that transfected macrophages secreted extracellular vesicles, exosomes, packed with catalase genetic material, pDNA and mRNA, active catalase, and NF-κb, a transcription factor involved in the encoded gene expression. Exosomes efficiently transfer their contents to contiguous neurons resulting in de novo protein synthesis in target cells. Thus, genetically modified macrophages serve as a highly efficient system for reproduction, packaging, and targeted gene and drug delivery to treat inflammatory and neurodegenerative disorders.

  1. Synthesis, characterization and transfection activity of new saturated and unsaturated cationic lipids.

    Science.gov (United States)

    Arpicco, Silvia; Canevari, Silvana; Ceruti, Maurizio; Galmozzi, Enrico; Rocco, Flavio; Cattel, Luigi

    2004-11-01

    We synthesized new cationic lipids, analogue to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), in order to compare those containing a dodecyl chain with those having a relatively long chain with two or five double bonds, such as squalenyl and dihydrofarnesyl derivatives, or complex saturated structures, such as squalane derivatives. The fusogenic helper lipid dioleoylphosphatidylethanolamine (DOPE) was added to cationic lipids to form a stable complex. Liposomes composed of 50:50 w/w cationic lipid/DOPE were prepared and incubated with plasmidic DNA at various charge ratios and the diameter and zeta potential of the complexes were measured. The surface charge of the DNA/lipid complexes can be controlled by adjusting the cationic lipid/DNA ratio. Finally, we tested the in vitro transfection efficiency of the cationic lipid/DNA complexes using different cell lines. The transfection efficiency was highest for the dodecyloxy derivative containing a single hydroxyethyl group in the head, followed by the dodecyloxy and the farnesyloxy trimethylammonium derivatives. Instead the C27 squalenyl and C27 squalanyl derivatives resulted inactive.

  2. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J. [and others

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  3. A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: preparation and gene transfection in vitro.

    Science.gov (United States)

    Ren, Jianli; Zhang, Ping; Tian, Ju; Zhou, Zhiyi; Liu, Xingzhao; Wang, Dong; Wang, Zhigang

    2014-09-01

    Targeted and high efficient gene delivery is a main issue in gene treatment. Taking advantage of ischemic memory target P-selectin and our previous study-synergistic effects of ultrasound-targeted microbubble destruction (UTMD) and TAT peptide on gene transfection, which were characterized by targeted aggregation and high efficient gene transfection, we set up a 'smart' gene delivery system-targeted ultrasound contrast agent (UCA) carrying gene and cell-permeable peptides (CPP). Such UCA had a strong binding force with DNA which was protected from being hydrolysed by nuclease. Moreover, synergistic effects of UTMD and TAT peptide increased gene transfection. Specifically, the UCA were reacted with an ischemic memory target P-selectin overexpressed by ischemic issues (including ischemic heart disease) and loaded with gene and CPP, which enabled targeted localization and gene delivery to ischemic cells overexpressing P-selectin. We demonstrated their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro. The results of confocal laser scanning microscopy (CLSM) showed that gene and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy, which demonstrated that the antibody was successfully connected to the UCA. The targeted UCA was specifically and tightly binded to hypoxia HUVEC, while there were no or little non-targeted UCA binding around hypoxia HUVEC. 24h after transfection, gene transfection efficiency detected by FCM was higher in targeted group than non-targeted group. Overall, the targeted UCA carrying gene and CPP was prepared successfully. It had a strong target binding capacity to hypoxia HUVEC and high efficient gene transfection, which maybe provide a novel strategy for gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Design of pH-sensitive peptides from natural antimicrobial peptides for enhancing polyethylenimine-mediated gene transfection.

    Science.gov (United States)

    Zhang, Shi-Kun; Song, Jin-Wen; Li, Su-Bo; Gao, Hong-Wei; Chang, Hong-Yu; Jia, Li-Li; Gong, Feng; Tan, Ying-Xia; Ji, Shou-Ping

    2017-05-01

    Poor endosomal release is a major barrier of polyplex-mediated gene transfection. Antimicrobial peptides (AMPs) are commonly used to improve polyethylenimine (PEI)-mediated gene transfection by increasing endosomal release. In the present study, we designed novel pH-sensitive peptides that highly enhance transfection efficiency compared to their parent peptides. Two analogues of melittin (Mel) and RV-23 (RV) were synthesized by replacing the positively-charged residues in their sequences with glutamic acid residues. The pH-sensitive lysis ability of the peptides, the effect of the peptides on physicochemical characteristics, the intracellular trafficking, the transfection efficiency, and the cytotoxicity of the polyplexes were determined. The acidic peptides showed pH-sensitive lytic activity. The hemolytic activity of acidic peptides at pH 5.0 was higher than that at pH 7.4. The incorporation of acidic peptides did not affect the DNA binding ability of PEI but affected the physicochemical characteristics of the PEI/DNA polyplexes, which may be beneficial for endosomal release and gene transfection. The incorporation of acidic peptides into PEI/DNA polyplexes enhanced the PEI-mediated transfection efficiency corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone. The results of the present study indicate that replacement of positively-charged residues with glutamic acid residues in the AMP sequence yields pH-sensitive peptides, which enhance the transfection efficiency of PEI/DNA polyplexes in various cell lines. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

    Science.gov (United States)

    Bhutto, Imran; Handa, James T.; Green, Jordan J.

    2012-01-01

    A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (ptransfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, ptransfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases. PMID:22629417

  6. The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release

    International Nuclear Information System (INIS)

    Kim, Bong Yoon; Sahara, Yoshinori; Yamamoto, Akitsugu; Kominami, Eiki; Kohsaka, Shinichi; Akazawa, Chihiro

    2006-01-01

    Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca 2+ -dependent exocytosis, which was abolished by the Ca 2+ -channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A

  7. Enhanced effect of nuclear localization signal peptide during ultrasound‑targeted microbubble destruction‑mediated gene transfection.

    Science.gov (United States)

    Cao, Sheng; Zhou, Qing; Chen, Jin-Ling; Jiang, Nan; Wang, Yi-Jia; Deng, Qing; Hu, Bo; Guo, Rui-Qiang

    2017-07-01

    Ultrasound‑targeted microbubble destruction (UTMD) can promote the entry of plasmid DNA (pDNA) into the cell cytoplasm, by increasing the permeability of the cell membrane. But the transfection efficiency remains low due to inability of the pDNA to enter the nucleus. Various methods have been explored to improve the UTMD transfection efficiency, but with little success. In cells, the classic nuclear localization signal (cNLS) peptide is an amino acid sequence that signals proteins that are due for nuclear transport. The present study aimed to investigate whether binding of a cNLS peptide to the pDNA may improve the transfection efficiency of UTMD. Four experimental groups were analyzed: Control group (UTMD + pDNA), group with cNLS (UTMD + pDNA + cNLS), group with mutated NLS (mNLS; UTMD + pDNA + mNLS), and group with cNLS and the nuclear import blocker, wheat germ agglutinin (WGA; UTMD + pDNA + cNLS + WGA). The NLS was labeled by fluorescein isothiocyanate, whereas pDNA was labeled with Cy3. Different molar ratios were tested for the NLS and pDNA combination in order to achieve optimal binding of the two molecules. Human umbilical vein endothelial cells were then transfected using the optimum ultrasonic irradiation parameters and NLS/pDNA molar ratio. At 6 h post‑transfection, the rates of Cy3‑labeled pDNA inside the cells and their nuclei were detected by flow cytometry and laser confocal microscopy, and the cellular vs. nuclear uptake of pDNA was calculated. In order to further evaluate the effect of NLS on UTMD‑mediated gene transfection, the transfection efficiency and relative expression levels of mRNA and protein were detected at 48 h post‑transfection. The results demonstrated that the optimal molar ratio of NLS with pDNA was 104:1. The rates of pDNA successful entry into the cell and nucleus were significantly higher in the cNLS group compared with the control group. The transfection efficiency, and relative expression levels of mRNA and protein

  8. [EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Xia, Delin; Huang, Mingke; Fu, Guangxing; Ma, Zheng; Wu, Shuangjiang; Zhou, Hangyu

    2015-01-01

    To study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents: BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2. 0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94 ± 0.12 in the experimental group, and was 4.99 ± 0.08 in the conventional transfection group, showing significant difference (t = 360.28, P = 0.02). The transfection efficiency was increased by 19% in the experimental group. 0.010% Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSUs, and can improve the transfection efficiency.

  9. Highly Branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate for High Performance Gene Transfection

    Directory of Open Access Journals (Sweden)

    Ming Zeng

    2017-05-01

    Full Text Available The top-performing linear poly(β-amino ester (LPAE, poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate (C32, has demonstrated gene transfection efficiency comparable to viral-mediated gene delivery. Herein, we report the synthesis of a ser