WorldWideScience

Sample records for included dna transfection

  1. 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.

  2. 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

  3. 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)

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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...

  9. 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.

  10. 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.

  11. 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)

  12. 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

  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. 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.

  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. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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)

  3. 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.

  4. 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

  5. Enhancing oligodendrocyte differentiation by transient transcription activation via DNA nanoparticle-mediated transfection.

    Science.gov (United States)

    Li, Xiaowei; Tzeng, Stephany Y; Zamboni, Camila Gadens; Koliatsos, Vassilis E; Ming, Guo-Li; Green, Jordan J; Mao, Hai-Quan

    2017-05-01

    Current approaches to derive oligodendrocytes from human pluripotent stem cells (hPSCs) need extended exposure of hPSCs to growth factors and small molecules, which limits their clinical application because of the lengthy culture time required and low generation efficiency of myelinating oligodendrocytes. Compared to extrinsic growth factors and molecules, oligodendrocyte differentiation and maturation can be more effectively modulated by regulation of the cell transcription network. In the developing central nervous system (CNS), two basic helix-loop-helix transcription factors, Olig1 and Olig2, are decisive in oligodendrocyte differentiation and maturation. Olig2 plays a critical role in the specification of oligodendrocytes and Olig1 is crucial in promoting oligodendrocyte maturation. Recently viral vectors have been used to overexpress Olig2 and Olig1 in neural stem/progenitor cells (NSCs) to induce the maturation of oligodendrocytes and enhance the remyelination activity in vivo. Because of the safety issues with viral vectors, including the insertional mutagenesis and potential tumor formation, non-viral transfection methods are preferred for clinical translation. Here we report a poly(β-amino ester) (PBAE)-based nanoparticle transfection method to deliver Olig1 and Olig2 into human fetal tissue-derived NSCs and demonstrate efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. This approach is potentially translatable for engineering stem cells to treat injured or diseased CNS tissues. Current protocols to derive oligodendrocytes from human pluripotent stem cells (hPSCs) require lengthy culture time with low generation efficiencies of mature oligodendrocytes. We described a new approach to enhance oligodendrocyte differentiation through nanoparticle-mediated transcription modulation. We tested an effective transfection method using cell-compatible poly (β-amino ester) (PBAE)/DNA nanoparticles as gene carrier to deliver

  6. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    Science.gov (United States)

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Data on macrophage mediated muscle transfection upon delivery of naked plasmid DNA with block copolymers

    Directory of Open Access Journals (Sweden)

    Vivek Mahajan

    2016-06-01

    Full Text Available The data contains 14 figures supporting the research article “Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers” [1]. The data explains the surgical procedure and histological characterization of Murine Hind Limb Ischemia. The data also shows the kinetics of luciferase gene expression, spread of GFP expression through muscle and the colocalization of GFP with cellular markers in ischemic muscles injected with pDNA alone or pDNA/Pluronic. Finally the data shows the effect of Pluronic Block Copolymer to enhance total gene expression (cmv-promoter driven luciferase gene in coculture of DNA transfected MØs with muscle cells.

  8. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    Science.gov (United States)

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  9. 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.

  10. Improved DNA condensation, stability, and transfection with alkyl sulfonyl-functionalized PAMAM G2

    Energy Technology Data Exchange (ETDEWEB)

    Rata-Aguilar, Azahara, E-mail: azahara@ugr.es; Maldonado-Valderrama, Julia; Jódar-Reyes, Ana Belén; Ortega-Vinuesa, Juan Luis [University of Granada, Biocolloid and Fluid Physics Group, Department of Applied Physics (Spain); Santoyo-Gonzalez, Francisco [University of Granada, Organic Chemistry Department, Institute of Biotechnology (Spain); Martín-Rodríguez, Antonio [University of Granada, Biocolloid and Fluid Physics Group, Department of Applied Physics (Spain)

    2015-04-15

    In this work, we have used a second-generation PAMAM grafted with octadecyl sulfonyl chains to condense plasmid DNA. The influence of this modification at different levels was investigated by comparison with original PAMAM G2. The condensation process and temporal stability of the complexes was studied with DLS, finding that the aliphatic chains influence DNA compaction via hydrophobic forces and markedly improve the formation and temporal stability of a single populated system with a hydrodynamic diameter below 100 nm. Interaction with a cell membrane model was also evaluated with a pendant drop tensiometer, resulting in further incorporation of the C18-PAMAM dendriplexes onto the interface. The improvement observed in transfection with our C18 grafted PAMAM is ascribed to the size, stability, and interfacial behavior of the complexes, which in turn are consequence of the DNA condensation process and the interactions involved.

  11. 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

  12. 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.

  13. 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.

  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. 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

  16. Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release

    Directory of Open Access Journals (Sweden)

    Zhang L

    2013-09-01

    Full Text Available Lifen Zhang,* Zhenzhen Chen,* Yanfeng LiState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology, Lanzhou University, Lanzhou, People's Republic of China*These authors contributed equally to this workAbstract: Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]–polyethyleneimine [PEI]–SS were synthesized through the addition of low molecular weight (800 Da PEI cross-linked with SS (PEI-SS to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3 with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers.Keywords: Pluronic, PEI, gene vector, dual-degradable, disulfide-containing linker

  17. Influence of DNA-Microbubble Coupling on Contrast Ultrasound-Mediated Gene Transfection in Muscle and Liver.

    Science.gov (United States)

    Xie, Aris; Wu, Melinda D; Cigarroa, Gabriella; Belcik, J Todd; Ammi, Azzdine; Moccetti, Federico; Lindner, Jonathan R

    2016-08-01

    Contrast ultrasound-mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver. Charge coupling of fluorescently labeled cDNA to either neutral MBs (MBN) or cationic MBs (MB+) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MBN or MB+ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 μg/10(8) MBs). Charge-coupling of cDNA was detected for MB+ but not MBN. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB+ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB+, which was approximately fivefold higher (P transfection with MBN equal to that of MB+. Efficient charge-coupling of cDNA to MB+ but not MBN occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake. Copyright © 2016 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.

  18. Construction of spherical liposome on solid transducers for electrochemical DNA sensing and transfection.

    Science.gov (United States)

    Bhuvana, Mohanlal; Dharuman, Venkataraman

    2014-10-01

    Cationic 1,2-dioleoyl trimethyl ammonium propane (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are anchored on cysteamine (cyst), mercaptopropionic acid (MPA) monolayer (thiol monolayers) modified on an individual gold transducer. DOTAP and DOPE are mixed with gold nanoparticle (AuNP) to form spherical liposome-AuNP. The electrochemical behaviors of the surface attached DOTAP-AuNP and DOPE-AuNP in presence of [Fe(CN)6](3-/4-) depend on the method of layer formation. Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and ultraviolet (UV)-visible spectroscopic techniques are used to characterize the liposome-AuNP nanocomposite. The studies indicate stability of spherical liposome-AuNP on the gold transducer. Label-free DNA hybridization detection on these surfaces reveals different detection limits. Confocal laser scanning microscopy (CLSM) is used to confirm the cell transfection.

  19. 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

  20. Apo B100 similarities to viral proteins suggest basis for LDL-DNA binding and transfection capacity.

    Science.gov (United States)

    Guevara, Juan; Prashad, Nagindra; Ermolinsky, Boris; Gaubatz, John W; Kang, Dongcheul; Schwarzbach, Andrea E; Loose, David S; Guevara, Natalia Valentinova

    2010-07-01

    LDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry.

  1. High efficiency transfection of embryonic limb mesenchyme with plasmid DNA using square wave pulse electroporation and sucrose buffer.

    Science.gov (United States)

    Bobick, Brent E; Alexander, Peter G; Tuan, Rocky S

    2014-01-01

    Micromass cultures of primary embryonic limb mesenchyme are a valuable model system for studying cartilage formation in vitro. However, high efficiency introduction of plasmid DNA into this hard-to-transfect cell type typically results in considerable cell death and significantly impeded chondrogenesis when the cells are subsequently plated in high density micromass. Here, we describe a novel method in which square wave pulse electroporation of chick embryo wing bud mesenchyme suspended in protective sucrose buffer results in high efficiency transfection without substantially affecting micromass culture cell viability or chondrogenic differentiation potential. Furthermore, we show that this protocol can be employed, along with effector gene expression vectors, to generate observable changes in the amount of cartilage tissue formed in micromass, unlike lower efficiency, higher cytotoxicity techniques that require co-transfection of reporter plasmids to monitor changes in the extent of chondrogenesis and correct for differences in cell viability.

  2. Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution, colloidal properties and DNA interaction

    Energy Technology Data Exchange (ETDEWEB)

    Steitz, Benedikt [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Hofmann, Heinrich [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Kamau, Sarah W. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Hassa, Paul O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zuerich, Zurich (Switzerland); Rechenberg, Brigitte von [Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich (Switzerland); Hofmann-Amtenbrink, Magarethe [MatSearch, Chemin Jean Pavillard 14, 1009 Pully (Switzerland); Petri-Fink, Alke [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland)]. E-mail: alke.fink@epfl.ch

    2007-04-15

    Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, {zeta}-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency.

  3. Effects of osteoprotegerin from transfection of pcDNA3.1(+/chOPG on bioactivity of chicken osteoclasts

    Directory of Open Access Journals (Sweden)

    Yao Jing

    2011-03-01

    Full Text Available Abstract Background Osteoprotegerin (OPG has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG expressed in chicken embryo fibroblasts (CEFs on chicken osteoclast function in vitro. Methods The chOPG sequence containing the open reading frame (ORF was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+ vector. PcDNA3.1 (+/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+ group and pcDNA3.1 (+/chOPG group. The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts. Results 48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the

  4. 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.

  5. 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...

  6. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    Directory of Open Access Journals (Sweden)

    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

  7. 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.

  8. 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

  9. Elucidating the interplay between DNA-condensing and free polycations in gene transfection through a mechanistic study of linear and branched PEI

    DEFF Research Database (Denmark)

    Dai, Zhuojun; Gjetting, Torben; Mattebjerg, Maria Ahlm

    2011-01-01

    In the present study we compare LPEI and BPEI characteristics related to DNA condensation and their role as free polycation chains in gene transfection. Using radioactive 32P labeled DNA, we investigated the effect of free PEI chains on the cellular uptake of polyplexes. Our investigations show...... different properties of BPEI and LPEI polyplexes in condensation and de-condensation processes as well as in cellular uptake, which was tightly correlated with transfection efficiency. In agreement with earlier reports we find all DNA to be condensed at N/P = 3. Further added PEI chains remain free...... in solution. We found that both the cellular uptake and gene transfection of BPEI polyplexes is much more efficient than LPEI polyplexes at a low N/P ratio of 3 (i.e., without free PEI chains). When N/P is high (10, with 7 portions of free PEI), the LPEI and BPEI polyplexes have similar transfection...

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid

    International Nuclear Information System (INIS)

    Green, M.H.L.; Lowe, J.E.

    1987-01-01

    Two SV40-transformed human fibroblast cell lines were transfected with plasmids in which double-strand breaks had been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cells. The apparent defect in transfection of AT5BIVA by pSV2gpt appeared to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. It is suggested that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. The results are also relevant to standard transfection experiments; they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime. (author)

  16. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

    Science.gov (United States)

    Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

    2017-02-01

    Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

  17. DNA transfection of bone marrow mesenchymal stem cells using micro electroporation chips

    KAUST Repository

    Deng, Peigang

    2011-02-01

    Experimental study of electroporation of bone marrow mesenchymal stem cells (MSCs) at the single-cell level was carried out on a micro EP chip by using single electric rectangular pulse. The threshold values of the electrode potential and pulse width for gas bubble generation on the micro electrodes due to electrolysis of water were revealed as 4.5 volt and 100 μs, respectively. Quantitative EP study was performed with various electric field strengths for various pulse widths, ranging from 20μs to 15ms. Over 1,000 single-cell EP results were used to construct an EP "phase diagram", which delineates the boundaries for (1) effective EP of MSCs and (2) electric cell lysis of MSCs. Finally, the micro EP chip showed successful transfection of the pEGFP-C1 plasmid into the MSCs by properly choosing the electric parameters from the EP "phase diagram". © 2011 IEEE.

  18. Lipid-based DNA/siRNA transfection agents disrupt neuronal bioenergetics and mitophagy.

    Science.gov (United States)

    Napoli, Eleonora; Liu, Siming; Marsilio, Ilaria; Zarbalis, Konstantinos; Giulivi, Cecilia

    2017-11-10

    A multitude of natural and artificial compounds have been recognized to modulate autophagy, providing direct or, through associated pathways, indirect entry points to activation and inhibition. While these pharmacological tools are extremely useful in the study of autophagy, their abundance also suggests the potential presence of unidentified autophagic modulators that may interfere with experimental designs if applied unknowingly. Here, we report unanticipated effects on autophagy and bioenergetics in neuronal progenitor cells (NPCs) incubated with the widely used lipid-based transfection reagent lipofectamine (LF), which induced mitochondria depolarization followed by disruption of electron transport. When NPCs were exposed to LF for 5 h followed by 24, 48, and 72 h in LF-free media, an immediate increase in mitochondrial ROS production and nitrotyrosine formation was observed. These events were accompanied by disrupted mitophagy (accumulation of dysfunctional and damaged mitochondria, and of LC3II and p62), in an mTOR- and AMPK-independent manner, and despite the increased mitochondrial PINK1 (PTEN-inducible kinase 1) localization. Evidence supported a role for a p53-mediated abrogation of parkin translocation and/or abrogation of membrane fusion between autophagosome and lysosomes. While most of the outcomes were LF-specific, only two were shared by OptiMEM exposure (with no serum and reduced glucose levels) albeit at lower extents. Taken together, our findings show that the use of transfection reagents requires critical evaluation with respect to consequences for overall cellular health, particularly in experiments designed to address autophagy-inducing effects and/or energy stress. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. Effect of nicotinic acid conjugated to DNA-transfecting complexes targeted at the transferrin receptor of HeLa cells.

    Science.gov (United States)

    Quick, G; van Zyl, J; Hawtrey, A; Ariatti, M

    2000-01-01

    A conjugate consisting of streptavidin (biotinylated transferrin)-biotinylated polylysine for DNA delivery to cells was modified by partial nicotinylation of the polylysine component of the conjugate and used for transfection studies. A conjugate of biotin10-nicotinyl60-polylysine250 containing 60 weakly basic nicotinyl (pyridine-3-carboxyl) residues was prepared. The design of the modified polylysine was directed to the possible binding of H+ ions in the endosome-lysosomal vesicles (pH 5-6) by the nicotinyl groups, thus circumventing the use of chloroquine. The results obtained, however, while showing a 5- to 6-fold increase in luciferase transfection activity still necessitated an absolute requirement for chloroquine. A further polylysine conjugate containing a larger number of nicotinyl residues, biotin10-nicotinyl120-polylysine250, also was prepared and studied. This macromolecule stimulated luciferase activity to a small extent and was also dependent on chloroquine. Smaller biotinylated polylysine100 conjugates containing nicotinyl groups were also prepared. These were biotin10-nicotinyl30-polylysine100, and biotin10-nicotinyl60-polylysine100, respectively. Both substances, however, gave opaque, hazy aqueous solutions with precipitates on standing and could not be used for further experimental work. The results indicate that the introduction of weakly basic nicotinyl (pyridine-3-carboxyl) groups onto polylysine250 give conjugates that are unable to replace the lysosomotrophic agent chloroquine in the HeLa cell sysem studied. A 5- to 6-fold increase in luciferase activity, however, was found with biotin10-nicotinyl60-polylysine250.

  20. Gene transfection of human mesenchymal stem cells with a nano-hydroxyapatite-collagen scaffold containing DNA-functionalized calcium phosphate nanoparticles.

    Science.gov (United States)

    Tenkumo, Taichi; Vanegas Sáenz, Juan Ramón; Takada, Yukyo; Takahashi, Masatoshi; Rotan, Olga; Sokolova, Viktoriya; Epple, Matthias; Sasaki, Keiichi

    2016-07-01

    This study aimed to fabricate a growth factor-releasing biodegradable scaffold for tissue regeneration. We prepared multishell calcium phosphate (CaP) nanoparticles functionalized with DNA, polyethyleneimine (PEI), protamine and octa-arginine (R8) and compared their respective transfection activity and cell viability measures using human mesenchymal stem cells. DNA-protamine complexes improved the transfection efficiency of CaP nanoparticles with the exception of those functionalized with R8. These complexes also greatly reduced the cytotoxicity of PEI. In addition, we also fabricated DNA-protamine-functionalized CaP nanoparticle-loaded nano-hydroxyapatite-collagen scaffolds and investigated their gene transfection efficiencies. These experiments showed that the scaffolds were associated with moderate hMSC cell viability and were capable of releasing the BMP-2 protein into hMSCs following gene transfection. In particular, the scaffold loaded with protamine-containing CaP nanoparticles showed the highest cell viability and transfection efficiency in hMSCs; thus, it might be suitable to serve as an efficient growth factor-releasing scaffold. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  1. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    International Nuclear Information System (INIS)

    Chowdhury, E.H.

    2011-01-01

    Highlights: → Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. → Fluoridated carbonate apatite promotes a robust increase in transgene expression. → Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  2. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  3. The role of the HCR system in the repair of lethal lesions of Bacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

    International Nuclear Information System (INIS)

    Vizdalova, M.; Janovska, E.; Zhestyanikov, V.D.

    1980-01-01

    The role of the HCR system in the repair of prelethal lesions induced by UV light, γ radiation and alkylating agents was studied in the Bacillus subtilis SPP1 phage, its heat sensitive mutants (N3, N73 nad ts 1 ) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher in hcr + cells than in hcr cells, the differences being more striking in intact phages than in their transfecting DNA's. Repair inhibitors reduce survival in hcr + cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growing hcr + cells but has no effect on its survival in competent hcr + cells; acriflavin and ethidium bromide decrease the survival of the UV-irradiated SPP1 phage in both exponentially growing and competent hcr + cells to the level of survival observed in hcr cells; moreover, ethidium bromide lowers the number of infective centres in hcr + cells of the UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of the UV-irradiated phages or their DNA in hcr cells. The repair mechanism under study also effectively repairs lesions induced by polyfunctional alkylating agents in the transfecting DNA's of B. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in the phages and their DNA by ethyl methanesulphonate or γ radiation. (author)

  4. In vivo Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

    Science.gov (United States)

    Shimizu, Kazunori; Kawakami, Shigeru; Hayashi, Kouji; Kinoshita, Hideyuki; Kuwahara, Koichiro; Nakao, Kazuwa; Hashida, Mitsuru; Konishi, Satoshi

    2012-01-01

    We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids. PMID:22844458

  5. In vivo site-specific transfection of naked plasmid DNA and siRNAs in mice by using a tissue suction device.

    Directory of Open Access Journals (Sweden)

    Kazunori Shimizu

    Full Text Available We have developed an in vivo transfection method for naked plasmid DNA (pDNA and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver. Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.

  6. Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

    Directory of Open Access Journals (Sweden)

    Alicia Martinez-Lopez

    Full Text Available The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV or viral haematopoietic septicaemia virus (VHSV, perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG₁₋₅₀₇ (the wild type membrane-anchored gpG of VHSV encoding either a soluble (gpG₁₋₄₆₂ or a secreted soluble (gpG(LmPle20-462 form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462 (the secreted soluble form conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in

  7. [Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene.].

    Science.gov (United States)

    Ye, Sujuan; Feng, Zhihua; Zhu, Wen; Cai, Chunji; Li, Lu; Sun, Liya; Wan, Haisu; Ma, Li; Zhou, Qinghua

    2008-08-20

    It has been proven that nm23-H1 gene is an important metastaticsuppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1 , we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH) in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1) by SSH method. The positive clones were preliminarily screened by bluewhite colony, and precisely identified by PCR. The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981). After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750) bp inserts. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981) are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  8. Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Jazayeri SD

    2013-02-01

    Full Text Available Seyed Davoud Jazayeri,1 Aini Ideris,1,2 Kamyar Shameli,3 Hassan Moeini,1 Abdul Rahman Omar1,21Institute of Bioscience, 2Faculty of Veterinary Medicine, 3Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, MalaysiaAbstract: In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV that induced cytokine expression, the hemagglutinin (H5 gene of AIV, A/Ck/Malaysia/5858/04 (H5N1 and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5 and formulated using green synthesis of silver nanoparticles (AgNPs with poly(ethylene glycol and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL-18, IL-15, and IL-12

  9. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression

    Science.gov (United States)

    Jaffri Masarudin, Mas; Yusoff, Khatijah; Rahim, Raha Abdul; Zobir Hussein, Mohd

    2009-01-01

    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO3- layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 Å in LDH to 42 Å was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.

  10. Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: Structure–Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations

    Science.gov (United States)

    2013-01-01

    Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure–activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6–10-fold over commercial reagents under their respective optimal conditions. Emerging structure–activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity. PMID:23682947

  11. Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

    Science.gov (United States)

    Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres

    2013-01-01

    Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells. PMID:23861978

  12. 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.

  13. Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene

    Directory of Open Access Journals (Sweden)

    Sujuan YE

    2008-08-01

    Full Text Available Background and objective It has been proven that nm23-H1 gene is an important metastatic-suppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1, we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. Methods The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1 by SSH method. The positive clones were preliminarily screened by blue-white colony, and precisely identified by PCR. Results The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981. After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750 bp inserts. Conclusion SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981 are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  14. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  15. Revisit complexation between DNA and polyethylenimine — Effect of length of free polycationic chains on gene transfection

    DEFF Research Database (Denmark)

    Yue, Yanan; Jin, Fan; Deng, Rui

    2011-01-01

    Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) r...

  16. Modulation of glutathione peroxidase expression by selenium: effect on human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells.

    OpenAIRE

    Chu, F F; Esworthy, R S; Akman, S; Doroshow, J H

    1990-01-01

    We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was charac...

  17. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Fujiyama, A.; Matsubara, K.

    1987-01-01

    A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes

  18. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

    Science.gov (United States)

    Tsurimoto, T; Fujiyama, A; Matsubara, K

    1987-01-01

    A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

  19. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    Science.gov (United States)

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

  20. [Construction and transfection of eucaryotic expression recombinant vector containing truncated region of UL83 gene of human cytomegalovirus and it's sheltered effect as DNA vaccine].

    Science.gov (United States)

    Gao, Rong-Bao; Li, Yan-Qiu; Wang, Ming-Li

    2006-06-01

    To construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human cytomegalovirus AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Results showed that quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 microg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was

  1. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels

    International Nuclear Information System (INIS)

    Cotten, M.; Laengle-Rouault, F.; Kirlappos, H.; Wagner, E.; Mechtler, K.; Zenke, M.; Beug, H.; Birnstiel, M.L.

    1990-01-01

    The authors have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, they have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle. They demonstrate here that this procedure is useful for a human leukemic cell line. They enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, they show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks transferrinfection, as does incubation of the cells at 18 degree C

  2. Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations

    Czech Academy of Sciences Publication Activity Database

    Reschel, Tomáš; Koňák, Čestmír; Oupický, D.; Seymour, L. W.; Ulbrich, Karel

    2002-01-01

    Roč. 81, 1-2 (2002), s. 201-217 ISSN 0168-3659 R&D Projects: GA ČR GV307/96/K226; GA AV ČR IAA1050101 Institutional research plan: CEZ:AV0Z4050913 Keywords : gene delivery * self assembly * polycation/DNA complexes Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.131, year: 2002

  3. Transfection of an expressive construct including IgG1 and Fv1 genes in ovary cell line for infliximab expression

    Directory of Open Access Journals (Sweden)

    Zohreh Sarabinejad

    2016-04-01

    Full Text Available Background: Infeliximab is a form of chimeric antibody which neutralizes the most important inflammatory cytokine, TNF-a, in inflammatory disorders. The aim of current study was to pilot expression of chimeric infliximab in Chinese Hamster ovary (CHO cells. Methods: In this research study, pVITRO2-neo-mcs vector that consist of infliximab light chain and heavy chain was used to transform into the E.coli by CaCl2 method. The plasmid was then purified and transfected to cultured CHO cells by Lipofectamine 2000® (Invitrogen GmbH, Germany. Transfected cells were selected upon G-418 treatment after 2 weeks and the level of expression, based on standard curve, was measured using IgG ELISA kit after 48 hours for each clone. High level expressed clone was then cultured in roller bottles and recombinant chimeric product was purified by protein A affinity chromatography. The purity of the product was analyzed by 10% gel SDS-PAGE from eluted samples. The efficacy of the purification was analyzed by ELISA before and after purification step. This article is a master's student thesis from February 2015 to August 2016 in pharmaceutical technology development center, Tehran University of Medical Sciences, Tehran, Iran. Results: The purified plasmid was analyzed on 2% agarose gel. After selective pressure of G-418, 10 stable transfect clones were assessed for infliximab secretion by IgG ELISA kit at 450 nm. The maximum and minimum expression which detected by ELISA were 23 ng/ml and 6 ng/ml, respectively. The band width of infliximab fraction during purification procedure was observed at 0.7-0.8 min. The efficiency of the purification by ELISA was 70%. On SDS-PAGE analysis, two bands, 25 and 50 kDa, respect to light and heavy chains of Infliximab, was confirmed the expression of recombinant protein. Conclusion: In the current study, the construct for infliximab monoclonal antibody production was designed using genetic engineering techniques and the expression

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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)

  10. 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.

  11. Polyethyleneimine mediated DNA transfection in schistosome parasites and regulation of the WNT signaling pathway by a dominant-negative SmMef2.

    Directory of Open Access Journals (Sweden)

    Shuang Liang

    Full Text Available Schistosomiasis is a serious global problem and the second most devastating parasitic disease following malaria. Parasitic worms of the genus Schistosoma are the causative agents of schistosomiasis and infect more than 240 million people worldwide. The paucity of molecular tools to manipulate schistosome gene expression has made an understanding of genetic pathways in these parasites difficult, increasing the challenge of identifying new potential drugs for treatment. Here, we describe the use of a formulation of polyethyleneimine (PEI as an alternative to electroporation for the efficacious transfection of genetic material into schistosome parasites. We show efficient expression of genes from a heterologous CMV promoter and from the schistosome Sm23 promoter. Using the schistosome myocyte enhancer factor 2 (SmMef2, a transcriptional activator critical for myogenesis and other developmental pathways, we describe the development of a dominant-negative form of the schistosome Mef2. Using this mutant, we provide evidence that SmMef2 may regulate genes in the WNT pathway. We also show that SmMef2 regulates its own expression levels. These data demonstrate the use of PEI to facilitate effective transfection of nucleic acids into schistosomes, aiding in the study of schistosome gene expression and regulation, and development of genetic tools for the characterization of molecular pathways in these parasites.

  12. 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.

  13. 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...

  14. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells.

    Science.gov (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan

    2016-09-01

    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. cDNA encoding a polypeptide including a hev ein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

    Science.gov (United States)

    Ogata, Jun; Uchiyama, Nahoko; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2013-04-10

    In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.

    Science.gov (United States)

    Khan, Mohammed A; Wu, Victoria M; Ghosh, Shreya; Uskoković, Vuk

    2016-06-01

    Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(l-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100×. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with

  16. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

    OpenAIRE

    Kaufman-Szymczyk, Agnieszka; Majewski, Grzegorz; Lubecka-Pietruszewska, Katarzyna; Fabianowska-Majewska, Krystyna

    2015-01-01

    Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscription...

  17. Microsatellite DNA reveals population genetic differentiation among sprat (Sprattus sprattus) sampled throughout the Northeast Atlantic, including Norwegian fjords

    DEFF Research Database (Denmark)

    Glover, Kevin A.; Skaala, Øystein; Limborg, Morten

    2011-01-01

    Glover, K. A., Skaala, Ø., Limborg, M., Kvamme, C., and Torstensen, E. Microsatellite DNA reveals population genetic differentiation among sprat (Sprattus sprattus) sampled throughout the Northeast Atlantic, including Norwegian fjords. – ICES Journal of Marine Science, 68: 2145–2151. Sprat (Sprat...... display population genetic differentiation throughout the Northeast Atlantic, and there may be limited connectivity between Norwegian fjord and sea-going populations.......Glover, K. A., Skaala, Ø., Limborg, M., Kvamme, C., and Torstensen, E. Microsatellite DNA reveals population genetic differentiation among sprat (Sprattus sprattus) sampled throughout the Northeast Atlantic, including Norwegian fjords. – ICES Journal of Marine Science, 68: 2145–2151. Sprat...

  18. 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...

  19. 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.

  20. 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...

  1. 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.

  2. 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.

  3. [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.

  4. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Agnieszka Kaufman-Szymczyk

    2015-12-01

    Full Text Available Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i post-translation histone modification (i.e., deacetylation and methylation; (ii DNA global hypomethylation; (iii promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

  5. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation.

    Science.gov (United States)

    Kaufman-Szymczyk, Agnieszka; Majewski, Grzegorz; Lubecka-Pietruszewska, Katarzyna; Fabianowska-Majewska, Krystyna

    2015-12-12

    Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

  6. 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)

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  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. A systematic study on Endotribelos Grodhaus (Diptera: Chironomidae) from Brazil including DNA barcoding to link males and females.

    Science.gov (United States)

    Trivinho-Strixino, Susana; Pepinelli, Mateus

    2015-03-18

    Six new species of Endotribelos from Brazil are described and illustrated as male, female, pupa and larva: E. bicolor sp. n., E. fulvidus sp. n., E. jaragua sp. n., E. jiboia sp. n., E. semibruneus sp. n. and E. sublettei sp. n. The female of E. calophylli Roque & Trivinho-Strixino and the larvae of four unknown morphotypes are also described. Keys including males and larvae of all known species of Endotribelos are provided. Adults' males and females from five species were linked using DNA Barcoding mtCOI sequences.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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...

  4. 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...

  5. Sequence Variation in the Ribosomal Internal Transcribed Spacers, Including the 5.8S rDNA, of Naegleria spp.

    Science.gov (United States)

    De Jonckheere, J F

    1998-09-01

    The ribosomal internal transcribed spacer (ITS), including the 5.8S rDNA, from the majority of the 11 described species of the amoeboflagellate Naegleria and from Willaertia magna have a size between 300 and 450 bp. In N. jadini and N. minor these products are approximately 750 bp long. The products from strains of the pathogenic N. fowleri vary between 323 and 423 bp. These length variations in N. fowleri are due to insertions of short repeats in the ITS1, causing the elongation of one stem-loop in the putative secondary structure. In all other species the sizes were identical from strains of the same species. In N. jadini and N. minor there are long inserts in the ITS2. Naegleria italica, N. clarki and N. galeacystis have shorter inserts in the ITS2. These inserts cause the elongation of one stem-loop in the putative secondary structure proposed for the ITS2. Because of the small differences in sequence between N. fowleri and N. lovaniensis the ITS does not provide target sequences for specifically identifying the pathogenic N. fowleri. However, differences in ITS1 do allow to distinguish different N. fowleri isolates. The ITS and 5.8S rDNA sequences will be of additional help in describing new Naegleria spp., which becomes more based on molecular data because morphological differences are scarce in these organisms. Copyright © 1998 Elsevier GmbH. All rights reserved.

  6. Induction of PLSCR1 in a STING/IRF3-Dependent Manner upon Vector Transfection in Ovarian Epithelial Cells

    Science.gov (United States)

    Kodigepalli, Karthik M.; Nanjundan, Meera

    2015-01-01

    Toll-like receptors (TLRs) are the primary sensors of the innate immune system that recognize pathogenic nucleic acids including double-stranded plasmid DNA (dsDNA). TLR signaling activates multiple pathways including IRF3 which is involved in transcriptional induction of inflammatory cytokines (i.e. interferons (IFNs)). Phospholipid scramblase 1, PLSCR1, is a highly inducible IFN-regulated gene mediating anti-viral properties of IFNs. Herein, we report a novel finding that dsDNA transfection in T80 immortalized normal ovarian surface epithelial cell line leads to a marked increase in PLSCR1 mRNA and protein. We also noted a comparable response in primary mammary epithelial cells (HMECs). Similar to IFN-2α treated cells, de novo synthesized PLSCR1 was localized predominantly to the plasma membrane. dsDNA transfection, in T80 and HMEC cells, led to activation of MAPK and IRF3. Although inhibition of MAPK (using U0126) did not modulate PLSCR1 mRNA and protein, IRF3 knockdown (using siRNA) significantly ablated the PLSCR1 induction. In prior studies, the activation of IRF3 was shown to be mediated by cGAS-STING pathway. To investigate the contribution of STING to PLSCR1 induction, we utilized siRNA to reduce STING expression and observed that PLSCR1 protein was markedly reduced. In contrast to normal T80/HMECs, the phosphorylation of IRF3 as well as induction of STING and PLSCR1 were absent in ovarian cancer cells (serous, clear cell, and endometrioid) suggesting that the STING/IRF3 pathway may be dysregulated in these cancer cells. However, we also noted induction of different TLR and IFN mRNAs between the T80 and HEY (serous epithelial ovarian carcinoma) cell lines upon dsDNA transfection. Collectively, these results indicate that the STING/IRF3 pathway, activated following dsDNA transfection, contributes to upregulation of PLSCR1 in ovarian epithelial cells. PMID:25658875

  7. Recombinant adeno-associated virus-, polyethylenimine/plasmid- and lipofectamine/carboxyfluorescein-labeled small interfering RNA-based transfection in retinal pigment epithelial cells with ultrasound and/or SonoVue.

    Science.gov (United States)

    Li, Hongli; Wan, Caifeng; Li, Fenghua

    2015-05-01

    The present study was conducted to investigate the efficacy and safety of ultrasound (US)‑mediated transfection of the type 2 recombinant adeno‑associated virus (AAV) vectors encoding the enhanced green fluorescent protein (EGFP) gene (rAAV), polyethylenimine (PEI)/plasmid EGFP‑N1 (pDNA) or lipofectamine (L)/carboxyfluorescein (FAM)‑labeled small interfering RNA (siRNA) in the human ARPE‑19 retinal pigment epithelial (RPE) cell line, with or without the addition of SonoVue. Cultured RPE cells were exposed to US, with or without SonoVue under different conditions, including variation in the intensity and duration of treatment, and the dose of microbubbles. The effects of ultrasound‑targeted microbubble destruction (UTMD) on the structure of pDNA and the transfection ability of rAAV, PEI/pDNA and L/siRNA were also evaluated. Furthermore, the effect of UTMD on RPE cells was evaluated at 0 and 24 h following UTMD. US‑mediated transfection (USMT) significantly increased L/siRNA transfection efficiency, as measured by the transgene expression per cell and the percentage of transfected cells. UTMD significantly increased rAAV and PEI/pDNA transfer to RPE cells. UTMD‑mediated rAAV or PEI/pDNA delivery was more effective than USMT‑mediated delivery of siRNA. Evaluating cell viability at 24 h post‑UTMD provided more valuable information than immediate evaluation following UTMD. Furthermore, there was minimal cytotoxicity and minimal change to the structure of pDNA under the optimal parameters. UTMD/US may be of use in enhancing rAAV, PEI/pDNA and L/siRNA transgene expression of ARPE‑19 cells in vitro. Studies on the transfection of different nucleotides (such as pDNA and siRNA) and different types of vectors (chemical and biological) mediated by UTMD may provide useful information to guide future in vivo and transfection studies.

  8. 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.

  9. 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.

  10. 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...

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. 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

  16. 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.

  17. The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation.

    Science.gov (United States)

    Uson, Maria Loressa; Ghosh, Shreya; Shuman, Stewart

    2017-09-01

    We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD + -dependent DNA ligases and especially by mycobacterial DNA ligases D and C. IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease. Copyright © 2017 American Society for Microbiology.

  18. 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.

  19. 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

  20. Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair.

    Science.gov (United States)

    Sinha, Krishna Murari; Stephanou, Nicolas C; Gao, Feng; Glickman, Michael S; Shuman, Stewart

    2007-05-18

    Mycobacterium tuberculosis and other bacterial pathogens have a Ku-dependent nonhomologous end joining pathway of DNA double-strand break repair. Here we identify mycobacterial UvrD1 as a novel interaction partner for Ku in a genome-wide yeast two-hybrid screen. UvrD1 per se is a vigorous DNA-dependent ATPase but a feeble DNA helicase. Ku stimulates UvrD1 to catalyze ATP-dependent unwinding of 3'-tailed DNAs. UvrD1, Ku, and DNA form a stable ternary complex in the absence of ATP. The Ku binding determinants are located in the distinctive C-terminal segment of UvrD1. A second mycobacterial paralog, UvrD2, is a vigorous Ku-independent DNA helicase. Ablation of UvrD1 sensitizes Mycobacterium smegmatis to killing by ultraviolet and ionizing radiation and to a single chromosomal break generated by I-SceI endonuclease. The physical and functional interactions of bacterial Ku and UvrD1 highlight the potential for cross-talk between components of nonhomologous end joining and nucleotide excision repair pathways.

  1. A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

    Science.gov (United States)

    Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

    2017-01-15

    In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection

  2. 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

  3. 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

  4. 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

  5. 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...

  6. 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

  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. 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.

  9. 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

  10. 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.

  11. 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

  12. Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

    Science.gov (United States)

    Corsaro, Daniele; Köhsler, Martina; Montalbano Di Filippo, Margherita; Venditti, Danielle; Monno, Rosa; Di Cave, David; Berrilli, Federica; Walochnik, Julia

    2017-04-01

    Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology.

    Science.gov (United States)

    Israelsson, S; Sävneby, A; Ekström, J-O; Jonsson, N; Edman, K; Lindberg, A M

    2014-12-01

    Oncolytic virotherapy is a promising novel form of cancer treatment, but the therapeutic efficiency needs improvement. A potential strategy to enhance the therapeutic effect of oncolytic viruses is to use infectious nucleic acid as therapeutic agent to initiate an oncolytic infection, without administrating infectious viral particles. Here we demonstrate improved viral replication activation efficiency when transfecting cells with 5' end authentic in vitro transcribed enterovirus RNA as compared to genomic RNA with additional non-genomic 5' nucleotides generated by conventional cloning methods. We used echovirus 5 (E5) as an oncolytoc model virus due to its ability to replicate in and completely destroy five out of six colon cancer cell lines and kill artificial colon cancer tumors (HT29 spheroids), as shown here. An E5 infectious cDNA clone including a hammerhead ribozyme sequence was used to generate in vitro transcripts with native 5' genome ends. In HT29 cells, activation of virus replication is approximately 20-fold more efficient for virus genome transcripts with native 5' genome ends compared to E5 transcripts generated from a standard cDNA clone. This replication advantage remains when viral progeny release starts by cellular lysis 22 h post transfection. Hence, a native 5' genomic end improves infection activation efficacy of infectious nucleic acid, potentially enhancing its therapeutic effect when used for cancer treatment. The clone design with a hammerhead ribozyme is likely to be applicable to a variety of oncolytic positive sense RNA viruses for the purpose of improving the efficacy of oncolytic virotherapy.

  18. DNA barcoding approaches for fishing authentication of exploited grouper species including the endangered and legally protected goliath grouper Epinephelus itajara

    Directory of Open Access Journals (Sweden)

    Rodrigo A. Torres

    2013-09-01

    Full Text Available Fishing strategies are constantly changing to meet the needs for new or alternative food sources. Consequently, management of fishing activities regarding rates of exploitation is essential, as a number of resources have reached situations of overexploitation. The aim of the present study was to use DNA barcoding from the goliath grouper and other exploited epinephelids in order to provide procedures for DNA authentication to be used as evidence for combating putative illegal fishing. The species studied were Epinephelus adscensionis, Mycteroperca bonaci, Mycteroperca interstitialis, Epinephelus itajara, Mycteroperca venenosa, Epinephelus mystacinus, Dermatolepis inermis, Alphestes afer, Cephalopholis fulva, Mycteroperca acutirostris, Rypticus saponaceus, Mycteroperca marginata and Epinephelus morio. Four of these species are the main epinephelids fished in the Atlantic Ocean. Differential patterns of polymerase chain reaction–restriction fragment length polymorphism were obtained from the species and additional single nucleotide polymorphisms were also detected among the four main epinephelids studied. The procedures proved very efficient and we suggest their applicability to the other fish groups as a way to control illegal capture and retail around the world, especially in cases in which filleting and other forms of de-characterization cause a lack of morpho-anatomical key characters.

  19. Geographic divergence of "Sulfolobus islandicus" strains assessed by genomic analyses including electronic DNA hybridization confirms they are geovars.

    Science.gov (United States)

    Zuo, Guanghong; Hao, Bailin; Staley, James T

    2014-02-01

    Ten well-annotated genomes of "Sulfolobus islandicus" strains from different geographic locations have been released at the NCBI database. Whole genome based composition vector trees indicate that these strains show the same branching patterns as originally reported by multi-locus sequence analysis. To determine whether the ten strains meet the criteria for separate species, DNA-DNA hybridization (DDH) was performed in silico. DDH values of strains from the same geographic location, i.e., Iceland, Kamchatka and North America, ranged from 82.4 to 95.4 %, clearly qualifying them as members of the same species. The lowest DDH values found between locations ranged from 75.5 to 76.6 %, which exceed the 70 % DDH threshold for a species thereby indicating they are all members of the same species based on the currently accepted definition. The clear divergences of strains from the different geographic locations are sufficiently great to consider them as separate geovars. "S. islandicus" has not yet been validly named and a type strain has not been deposited in culture collections. We urgently recommend that those who study the organism fulfill the criteria of the International Code of Nomenclature of Bacteria in order to designate a type strain and to identify and deposit related strains of this species to make them available to the broader scientific community.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes.

    Science.gov (United States)

    Oroojalian, Fatemeh; Rezayan, Ali Hossein; Shier, Wayne Thomas; Abnous, Khalil; Ramezani, Mohammad

    2017-05-15

    Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI 25 (25kDa PEI) or carboxyalkylated-PEI 10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143-173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI 25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI 10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity. Copyright © 2017

  6. Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies.

    Science.gov (United States)

    Mann, Anita; Thakur, Garima; Shukla, Vasundhara; Singh, Anand Kamal; Khanduri, Richa; Naik, Rangeetha; Jiang, Yang; Kalra, Namita; Dwarakanath, B S; Langel, Ulo; Ganguli, Munia

    2011-10-03

    Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

  7. EFFECTOR OF TRANSCRIPTION2 is involved in xylem differentiation and includes a functional DNA single strand cutting domain.

    Science.gov (United States)

    Ivanov, Rumen; Tiedemann, Jens; Czihal, Andreas; Schallau, Anna; Diep, Le Hong; Mock, Hans-Peter; Claus, Bernhard; Tewes, Annegret; Bäumlein, Helmut

    2008-01-01

    EFFECTORS OF TRANSCRIPTION2 (ET) are plant-specific regulatory proteins characterized by the presence of two to five C-terminal DNA- and Zn-binding repeats, and a highly conserved cysteine pattern. We describe the structural characterization of the three member Arabidopsis thaliana ET gene family and reveal some allelic sequence polymorphisms. A mutation analysis showed that AtET2 affects the expression of various KNAT genes involved in the maintenance of the undifferentiated state of cambial meristem cells. It also plays a role in the regulation of GA5 (gibberellin 3-beta-dioxygenase) and the cell-cycle-related GASA4. A correlation was established between AtET2 expression and the cellular differentiation state. AtET-GFP fusion proteins shuttle between the cytoplasm and nucleus, with the AtET2 product prevented from entering the nucleus in non-differentiating cells. Within the nucleus, AtET2 probably acts via a single strand cutting domain. A more general regulatory role for ET factors is proposed, governing cell differentiation in cambial meristems, a crucial process for the development of plant vascular tissues.

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

    Indian Academy of Sciences (India)

    1nanoTherics Limited, Med IC4, Keele University Science and Business Park, Keele, Staffordshire, ST5 5NL, UK. 2J. Crayton Pruitt Family ... lying the cell culture plate that pulls the complexes onto the surface of the cells (Plank et al. 2003 .... netic arrays play an important role in the internalization of magnetic nanoparticles ...

  9. Gene transfection using lipid-mediated TGFβ1 sense and antisense gene expression vectors and its effects on TGFβ1 and procollagen I mRNA expression in 60Co-irradiated human embryo lung fibroblasts

    International Nuclear Information System (INIS)

    Liu Chunjie; Wang Dewen; Zhang Zhaoshan

    2001-01-01

    Objective: To investigate the effects on gene expression of 60 Co-irradiated human embryo lung fibroblasts after gene transfection using lipid-mediated TGFβ1 sense and antisense gene expression vectors. Methods: TGFβ1 sense and antisense gene expression vectors were transfected using a lipid-mediated method. Gene expression was analysed by RNA dot blot. Results: HELFs irradiated with 5 Gy were transfected with an expression vector encoding the human TGFβ1 sense or antisense gene under control of the mouse mammary tumor virus long terminal repeat(MMTV-LTR) promoter/enhance sequence (pMAMneo-TGFβ1, or pMAMneo-anti-TGFβ1). The transfected cells elected by G418 resistance were cultured in DMEM containing dexamethasone. The chromosomal DNA and RNA were extracted. Positive reaction was showed from chromosomal DNA by a PCR method of neo-specific primers and DNA dot blot with Dig-labelling neo-specific probe. RNA dot blot analysis showed that TGFβ1 mRNA level of the cells transfected with pMAM neo-anti TGFβ1 decreased, but that of transfected with pMAM neo-TGFβ1 increasing. For procollagen I mRNA, the transfected pMAM neo-anti TGFβ1 was lower than un-transfected cells and the transfected pMAM neo-TGFβ1 was higher. Conclusion: After TGFβ1 sense and antisense gene transfection, TGFβ1 mRNA level of the cells transfected with TGFβ1 antisense gene decreased, but that with TGFβ1 sense gene increased. For procollagen I mRNA, the cells transfected with TGFβ1 antisense gene was lower than un-transfected cells and the cells transfected with TGFβ1 sense gene was higher than un-transfected cells

  10. 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

  11. 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.

  12. 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.

  13. 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...

  14. 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...

  15. 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 series of highly branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate (HC32 and explore how the branching structure influences the performance of C32 in gene transfection. HC32 were synthesized by an “A2 + B3 + C2” Michal addition strategy. Gaussia luciferase (Gluciferase and green fluorescent protein (GFP coding plasmid DNA were used as reporter genes and the gene transfection efficiency was evaluated in human cervical cancer cell line (HeLa and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK cells. We found that the optimal branching structure led to a much higher gene transfection efficiency in comparison to its linear counterpart and commercial reagents, while preserving high cell viability in both cell types. The branching strategy affected DNA binding, proton buffering capacity and degradation of polymers as well as size, zeta potential, stability, and DNA release rate of polyplexes significantly. Polymer degradation and DNA release rate played pivotal parts in achieving the high gene transfection efficiency of HC32-103 polymers, providing new insights for the development of poly(β-amino esters-based gene delivery vectors.

  16. Tracking the direct impact of rainfall on groundwater at Mt. Fuji by multiple analyses including microbial DNA

    Science.gov (United States)

    Sugiyama, Ayumi; Masuda, Suguru; Nagaosa, Kazuyo; Tsujimura, Maki; Kato, Kenji

    2018-02-01

    the heavy rain transported archaeal particles from the geologic layer along the groundwater route. This finding was supported by changes in constituents of Archaea, dominated by Halobacteriales and Methanobacteriales, which were not seen from other observations. Those two groups of Archaea are believed to be relatively tightly embedded in the geologic layer and were extracted from the environment to the examined groundwater through enforced piston flow. Microbial DNA can thus give information about the groundwater route, which may not be shown by analysis of chemical materials dissolved in the groundwater.

  17. 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.

  18. 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.

  19. Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

    Science.gov (United States)

    Roelse, Margriet; Henquet, Maurice G L; Verhoeven, Harrie A; de Ruijter, Norbert C A; Wehrens, Ron; van Lenthe, Marco S; Witkamp, Renger F; Hall, Robert D; Jongsma, Maarten A

    2018-02-16

    Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

  20. 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.

  1. Tracking the direct impact of rainfall on groundwater at Mt. Fuji by multiple analyses including microbial DNA

    Directory of Open Access Journals (Sweden)

    A. Sugiyama

    2018-02-01

    after the event. This suggests that strengthened piston flow caused by the heavy rain transported archaeal particles from the geologic layer along the groundwater route. This finding was supported by changes in constituents of Archaea, dominated by Halobacteriales and Methanobacteriales, which were not seen from other observations. Those two groups of Archaea are believed to be relatively tightly embedded in the geologic layer and were extracted from the environment to the examined groundwater through enforced piston flow. Microbial DNA can thus give information about the groundwater route, which may not be shown by analysis of chemical materials dissolved in the groundwater.

  2. 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.

  3. Mitochondrial DNA sequence evolution and phylogeny of the Atlantic Alcidae, including the extinct great auk (Pinguinus impennis).

    Science.gov (United States)

    Moum, Truls; Arnason, Ulfur; Arnason, Einar

    2002-09-01

    The Atlantic auk assemblage includes four extant species, razorbill (Alca torda), dovekie (Alle alle), common murre (Uria aalge), and thick-billed murre (U. lomvia), and one recently extinct species, the flightless great auk (Pinguinus impennis). To determine the phylogenetic relationships among the species, a contiguous 4.2-kb region of the mitochondrial genome from the extant species was amplified using PCR. This region included one ribosomal RNA gene, four transfer RNA genes, two protein-coding genes, the control region, and intergenic spacers. Sets of PCR primers for amplifying the same region from great auk were designed from sequences of the extant species. The authenticity of the great auk sequence was ascertained by alternative amplifications, cloning, and separate analyses in an independent laboratory. Phylogenetic analyses of the entire assemblage, made possible by the great auk sequence, fully resolved the phylogenetic relationships and split it into two primary lineages, Uria versus Alle, Alca, and Pinguinus. A sister group relationship was identified between Alca and Pinguinus to the exclusion of ALLE: Phylogenetically, the flightless great auk originated late relative to other divergences within the assemblage. This suggests that three highly divergent species in terms of adaptive specializations, Alca, Alle, and Pinguinus, evolved from a single lineage in the Atlantic Ocean, in a process similar to the initial adaptive radiation of alcids in the Pacific Ocean.

  4. Incorporation of DOPE into Lipoplexes formed from a Ferrocenyl Lipid leads to Inverse Hexagonal Nanostructures that allow Redox-Based Control of Transfection in High Serum.

    Science.gov (United States)

    Muller, John P E; Aytar, Burcu S; Kondo, Yukishige; Lynn, David M; Abbott, Nicholas L

    2012-01-01

    We report small angle X-ray and neutron scattering measurements that reveal that mixtures of the redox-active lipid bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) and dioleoylphosphatidylethanolamine (DOPE) spontaneously form lipoplexes with DNA that exhibit inverse hexagonal nanostructure (H(II) (c)). In contrast to lipoplexes of DNA and BFDMA only, which exhibit a multilamellar nanostructure (L(α) (c)) and limited ability to transfect cells in the presence of serum proteins, we measured lipoplexes of BFDMA and DOPE with the H(II) (c) nanostructure to survive incubation in serum and to expand significantly the range of media compositions (e.g., up to 80% serum) over which BFDMA can be used to transfect cells with high efficiency. Importantly, we also measured the oxidation state of the ferrocene within the BFDMA/DNA lipoplexes to have a substantial influence on the transfection efficiency of the lipoplexes in media containing serum. Specifically, whereas lipoplexes of reduced BFDMA and DOPE transfect cells with high efficiency, lipoplexes of oxidized BFDMA and DNA lead to low levels of transfection. Complementary measurements using SAXS reveal that the low transfection efficiency of the lipoplexes of oxidized BFDMA and DOPE correlates with the presence of weak Bragg peaks and thus low levels of H(II) (c) nanostructure in solution. Overall, these results provide support for our hypothesis that DOPE-induced formation of the H(II) (c) nanostructure of the BFDMA-containing lipoplexes underlies the high cell transfection efficiency measured in the presence of serum, and that the oxidation state of BFDMA within lipoplexes with DOPE substantially regulates the formation of the H(II) (c) nanostructure and thus the ability of the lipoplexes to transfect cells with DNA. More generally, the results presented in this paper suggest that lipoplexes formed from BFDMA and DOPE may offer the basis of approaches that permit active and external control of transfection of

  5. 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.

  6. Diversity of Y-chromosomal and mtDNA Markers Included in Mediscope Chip within Two Albanian Subpopulations from Croatia and Kosovo: Preliminary Data.

    Science.gov (United States)

    Čoklo, Miran; Auguštin, Dubravka Havaš; Šarac, Jelena; Novokmet, Natalija; SIndik, Joško; Lewis, Ana Perinić; Petranovic, Mateja Zajć; Mulahasanović, Lejla Kovačević; Khusnutdinova, Elza; Litvinov, Serghey; Haydar, Sara; Lautier, Corinne; Normand, Christophe; Attaoua, Redha; Vintila, Madalina; Bosch-Comas, Anna; Suarez, Helena; Jares, Pedro; Gomis, Ramon; Missoni, Saša; Marjanović, Damir; Grigorescu, Florin

    2016-09-01

    The aim of this preliminary study is to analyze genetic specificity of Kosovo Albanians comparing with neighboring populations using new genetic tool - MEDISCOPE gene chip, to investigate the feasibility of this approach. We collected 37 DNA samples (9 Croats, 17 Albanians from Croatia and 11 Albanians from Kosovo) from unrelated males born in Croatia and Kosovo. Additionally, samples were expanded with female individuals and mtDNA analysis included a total of 61 samples (15 Croats, 23 Albanians from Croatia and 23 Albanians from Kosovo). This pilot study suggests that the usage of the MEDISCOPE chip could be recognized as an efficient tool within recognition of the population genetic specificity even within extremely small sample size.

  7. An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system.

    Science.gov (United States)

    Hu, Q; Henney, H R

    1997-03-20

    We have isolated and sequenced a 2388 bp polyubiquitin encoding genomic DNA from Acanthamoeba encompassing two complete and one incomplete ubiquitin units. Codon usage frequency shows extreme bias. The deduced amino acid sequences of each unit are identical to each other and the same as that deduced from a previously sequenced Acanthamoeba castellanii cDNA. The upstream region of this gene, which contained some putative regulatory modules, was recovered by PCR (polymerase chain reaction) amplification and subcloning. This upstream fragment was ligated to the CAT (chloramphenicol acetyltransferase) gene in a eukaryotic expression plasmid and successfully applied to the establishment of an Acanthamoeba transient transfection system. Transfection was performed by electroporation and the optimal voltage was 4500 volts/cm at capacitance 25 microF. DEAE-dextran (25 microg/ml) added into the electroporation buffer increased the transfection efficiency by about 45%. The CAT activity was proportional to the amount of DNA transfected and reached the peak level 48 h after transfection. CAT assays showed that the polyubiquitin gene upstream fragment contains a functional promoter which is about 2.5 times as strong as a viral RSV-LTR promoter when driving CAT expression in Acanthamoeba.

  8. 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.

  9. Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

    Directory of Open Access Journals (Sweden)

    Adriana Cheavegatti-Gianotto

    2018-03-01

    Full Text Available Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis. The event also expresses neomycin-phosphotransferase type II (NptII protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as “pure substances, chemically defined,” by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as “GMO derivatives” and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein

  10. 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.

  11. 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

  12. 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)

  13. 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.

  14. 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.

  15. 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.

  16. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    Science.gov (United States)

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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.

  19. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    DEFF Research Database (Denmark)

    Yang, Hsiao Yin; Vonk, Lucienne A.; Licht, Ruud

    2014-01-01

    The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical...... application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20...... 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for si...

  20. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

    International Nuclear Information System (INIS)

    LingNah Su; Little, J.B.

    1992-01-01

    A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

  1. 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.

  2. 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%.

  3. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

    Science.gov (United States)

    Hujaya, Sry D; Marchioli, Giulia; Roelofs, Karin; van Apeldoorn, Aart A; Moroni, Lorenzo; Karperien, Marcel; Paulusse, Jos M J; Engbersen, Johan F J

    2015-05-10

    Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer

  4. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    Science.gov (United States)

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

    DEFF Research Database (Denmark)

    Glahder, Jacob; Norrild, Bodil; Persson, Mikael B

    2005-01-01

    Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and puls...

  6. Low-frequency ac electroporation shows strong frequency dependence and yields comparable transfection results to dc electroporation.

    Science.gov (United States)

    Zhan, Yihong; Cao, Zhenning; Bao, Ning; Li, Jianbo; Wang, Jun; Geng, Tao; Lin, Hao; Lu, Chang

    2012-06-28

    Conventional electroporation has been conducted by employing short direct current (dc) pulses for delivery of macromolecules such as DNA into cells. The use of alternating current (ac) field for electroporation has mostly been explored in the frequency range of 10kHz-1MHz. Based on Schwan equation, it was thought that with low ac frequencies (10Hz-10kHz), the transmembrane potential does not vary with the frequency. In this report, we utilized a flow-through electroporation technique that employed continuous 10Hz-10kHz ac field (based on either sine waves or square waves) for electroporation of cells with defined duration and intensity. Our results reveal that electropermeabilization becomes weaker with increased frequency in this range. In contrast, transfection efficiency with DNA reaches its maximum at medium frequencies (100-1000Hz) in the range. We postulate that the relationship between the transfection efficiency and the ac frequency is determined by combined effects from electrophoretic movement of DNA in the ac field, dependence of the DNA/membrane interaction on the ac frequency, and variation of transfection under different electropermeabilization intensities. The fact that ac electroporation in this frequency range yields high efficiency for transfection (up to ~71% for Chinese hamster ovary cells) and permeabilization suggests its potential for gene delivery. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. 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

  8. 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

  9. [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.

  10. 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.

  11. 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.

  12. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone

    International Nuclear Information System (INIS)

    Adachi, A.; Gendelman, H.E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M.A.

    1986-01-01

    The authors considered an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified

  13. 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.

  14. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

    Science.gov (United States)

    Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

    2015-09-01

    -stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Increased radiosensitivity of p16 gene-deleted human glioma cells after transfection with wild-type p16 gene

    International Nuclear Information System (INIS)

    Miyakoshi, Junji; Kitagawa, Kaori; Yamagishi, Nobuyuki; Ohtsu, Shuji; Takebe, Hikaru; Day, R.S. III.

    1997-01-01

    The A1235 and T98 cell lines derived from human gliomas have homozygous deletions in their p16 genes and are radiosensitive and radioresistant, respectively, with respect to other established glioma cell lines. These differences in radiosensitivity may be due to variations to some extent among cell lines, rather than genetically defined resistance or sensitivity. We examined the effect on radiation sensitivity of introducing a wild-type p16 gene into both p16-deficient glioma cell lines. The plasmid pOPMTS containing human wild-type p16 cDNA and a neomycin resistance gene, or the control plasmid pOPRSV1, were transfected into these cells. Clones from both cell lines, which expressed wild-type p16 mRNA constitutively after transfection with pOPMTS, were more radiosensitive than the parental cells and clones obtained after transfection with the negative control plasmid. (author)

  2. 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.

  3. Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands

    Directory of Open Access Journals (Sweden)

    Lindy L. Esterhuizen

    2012-09-01

    Full Text Available The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component or bipartite (two circular ssDNA components called DNA-A and DNA-B, many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-as or betasatellites (DNA-βs. Additionally, subgenomic molecules, also known as defective interfering (DIs DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.

  4. 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.

  5. Morphology and SSU rDNA sequence analysis of two hypotrichous ciliates (Protozoa, Ciliophora, Hypotrichia) including the new species Metaurostylopsis parastruederkypkeae n. sp.

    Science.gov (United States)

    Lu, Borong; Wang, Chundi; Huang, Jie; Shi, Yuhong; Chen, Xiangrui

    2016-10-01

    The morphology and phylogeny of two hypotrichous ciliates, Metaurostylopsis parastruederkypkeae n. sp. and Neourostylopsis flavicana (Wang et al., 2011) Chen et al., 2013 were investigated based on morphology, infraciliature and the small subunit (SSU) ribosomal RNA gene (rRNA) sequence. The new species, M. parastruederkypkeae n. sp. was identified according to its characteristics: body shape ellipsoidal, size about (165-200) × (45-60) μm in vivo, cell color reddish; two types of cortical granules including wheat grain-like and yellow-greenish larger ones along the marginal cirri rows and dorsal kineties and dot-like and reddish smaller ones, grouped around marginal cirri on ventral side and arranged in short lines on dorsal side; 26-41 adoral membranelles; three frontal and one parabuccal, five to seven frontoterminal, one buccal, and three to six transverse cirri; seven to thirteen midventral pairs; five to nine unpaired ventral cirri, five to seven left and three to five right marginal rows; and three complete dorsal kineties. Phylogenetic analysis based on SSU rDNA sequences showed that both Metaurostylopsis and Neourostylopsis are monophyletic. As the internal relationship between and within both genera are not clear, further studies on the species in these two genera are necessary. The key characteristics of all known twelve Metaurostylopsis-Apourostylopsis-Neourostylopsis species complex were updated.

  6. Faecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular ssDNA viruses.

    Science.gov (United States)

    Lima, Diane A; Cibulski, Samuel P; Finkler, Fabrine; Teixeira, Thais F; Varela, Ana Paula M; Cerva, Cristine; Loiko, Márcia R; Scheffer, Camila M; Dos Santos, Helton F; Mayer, Fabiana Q; Roehe, Paulo M

    2017-04-01

    This study is focused on the identification of the faecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with blastx revealed that 279 contigs (4 %) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding DNA viruses were also identified. The characterization of the faecal virome of healthy chickens described here not only provides a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

  7. 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.

  8. 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.

  9. The effects of human TSH receptor gene transfection on iodide uptake and thyroid-specific gene expression in poorly differentiated thyroid carcinoma cell line

    International Nuclear Information System (INIS)

    Hou Shasha; Wang Hui; Feng Fang; Lin Ning; Fu Hongliang; Du Xueliang; Wu Jingchuan

    2011-01-01

    Objective: To investigate the changes of iodide uptake and the expression of thyroid-specific genes in poorly differentiated follicular thyroid carcinoma (FTC) cells after transfection of human TSH receptor (hTSHR) gene in vitro. Methods: The recombinant eukaryotic expression plasmid PcDNA3.1/hTSHR-cDNA was transformed into DH 5a bacterial for amplification and then the recombinant plasmid was extracted. The recombinant was identified with PCR amplifying, restriction enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1/hTSHR was transfected into FTC-133 cell line by lipofectin method in vitro. Immunofluorescence, iodide uptake studies and real time-PCR were applied to detect target protein expression. Statistical analysis was performed with t-test using SPSS 13.0 software. Results: Kpn I and Xba I restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3.1/hTSHR was successfully constructed. After transfection of the recombinant plasmid pcDNA3.1/hTSHR-cDNA and the stimulation of hTSH, the tumor cells displayed the expression of hTSHR protein at cell surface and cytoplasm. The iodine uptake in pcDNA3.1/hTSHR transfected cells was 2.9 times higher than that of control(pcDNA3.1(+) transfected cells) group(t = 28.63, P<0.01). The expression of TSHR, NIS, TPO and Tg (mRNA levels) in pcDNA3.1/hTSHR transfected cells were also significantly elevated by 1.74 (t =5.959, P<0.01), 7.2 (t =3.807, P<0.05), 2.88 (t=4.769, P<0.01) and 2.67 times (t=6.388, P<0.01) respectively compared to those of the control group. Conclusion: The study demonstrates that iodide uptake may be reactivated by hTSHR receptor gene transfection in poorly differentiated FTC cell. (authors)

  10. The effect of H-ras oncogene transfection on response of mink lung epithelial cells to growth factors and cytotoxic drugs.

    Science.gov (United States)

    Kerr, D I; Plumb, J A; Freshney, R I; Khan, M Z; Spandidos, D A

    1991-01-01

    Mink lung epithelial cells were transfected with c-myc and activated H-ras genes. The transfected sublines formed colonies in soft agar and were tumorigenic when injected subcutaneously into athymic nude mice. DNA synthesis was measured in each of the cell lines by 3H-thymidine incorporation and in the parent line there was dose related stimulation of DNA synthesis by epidermal growth factor (EGF) and inhibition by transforming growth factor-beta (TGF-beta). The c-myc transfected line had a reduced inhibitory response to TGF-beta and an exaggerated stimulatory response to EGF whereas the activated H-ras1 transfected line did not respond to TGF-beta or EGF. The activated H-ras1 transfected line was significantly more resistant to doxorubicin (ID50, 4.4 nM) and vincristine (ID50, 4.9 nM) than the parent mink lung epithelial cell line (ID50, 2.7 nM and 2.4 nM respectively). It would appear that oncogene transfection can alter the sensitivity of mink lung epithelial cells to both exogenous growth factors and cytotoxic drugs.

  11. 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...

  12. 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...

  13. DNA copy number analysis of Grade II-III and Grade IV gliomas reveals differences in molecular ontogeny including chromothripsis associated with IDH mutation status.

    Science.gov (United States)

    Cohen, Adam; Sato, Mariko; Aldape, Kenneth; Mason, Clinton C; Alfaro-Munoz, Kristin; Heathcock, Lindsey; South, Sarah T; Abegglen, Lisa M; Schiffman, Joshua D; Colman, Howard

    2015-06-20

    Isocitrate dehydrogenase (IDH) mutation status and grade define subgroups of diffuse gliomas differing based on age, tumor location, presentation, and prognosis. While some biologic differences between IDH mutated (IDH (mut)) and wild-type (IDH (wt)) gliomas are clear, the distinct alterations associated with progression of the two subtypes to glioblastoma (GBM, Grade IV) have not been well described. We analyzed copy number alterations (CNAs) across grades (Grade II-III and GBM) in both IDH (mut) and IDH (wt) infiltrating gliomas using molecular inversion probe arrays. Ninety four patient samples were divided into four groups: Grade II-III IDH (wt) (n = 17), Grade II-III IDH (mut) (n = 28), GBM IDH (wt) (n = 25), and GBM IDH (mut) (n = 24). We validated prior observations that IDH (wt) GBM have a high frequency of chromosome 7 gain (including EGFR) and chromosome 10 loss (including PTEN) compared with IDH (mut) GBM. Hierarchical clustering of IDH (mut) gliomas demonstrated distinct CNA patterns distinguishing lower grade gliomas versus GBM. However, similar hierarchical clustering of IDH (wt) gliomas demonstrated no CNA distinction between lower grade glioma and GBM. Functional analyses showed that IDH (wt) gliomas had more chromosome gains in regions containing receptor tyrosine kinase pathways. In contrast, IDH (mut) gliomas more commonly demonstrated amplification of cyclins and cyclin dependent kinase genes. One of the most common alterations associated with transformation of lower grade to GBM IDH (mut) gliomas was the loss of chromosomal regions surrounding PTEN. IDH (mut) GBM tumors demonstrated significantly higher levels of overall CNAs compared to lower grade IDH (mut) tumors and all grades of IDH (wt) tumors, and IDH (mut) GBMs also demonstrated significant increase in incidence of chromothripsis. Taken together, these analyses demonstrate distinct molecular ontogeny between IDH (wt) and IDH (mut) gliomas. Our data also support the novel

  14. Effect of ionizing radiation on DNA-mediated gene transfer efficiency

    International Nuclear Information System (INIS)

    Rubin, J.S.; Hall, E.J.; Hei, T.K.

    1986-01-01

    Ionizing radiation causes a number of molecular changes in cells including DNA damage and gene amplification. In this study the authors examined whether radiation can effect the efficiency of integration and expression of exogenous DNA sequences. They examined both 137 Cs γ rays and various monoenergetic neutron beams. This enabled them to test whether the LET or RBE of the radiation had any effect. Rat2 cells were transfected with various amounts of the bacterial plasmid pSV2-GPT along with carrier DNA for 24 hours

  15. 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.

  16. Low molecular weight polyethylenimine-grafted soybean protein gene carriers with low cytotoxicity and greatly improved transfection in vitro.

    Science.gov (United States)

    Yao, Weijing; Cheng, Xu; Fu, Shengxiang; Yan, Guoqing; Wang, Xin; Tang, Rupei

    2018-02-01

    A series of gene carriers (SP-PEI) have been synthesized by acylation reaction between soybean protein and branched polyethylenimine with low molecular weight of 600, 1200 and 1800 Da, and designed as SP-PEI600, SP-PEI1200 and SP-PEI1800, respectively. SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes with size range of 100-200 nm, and exhibited much lower cytotoxicity against 293T and SH-SY5Y cells than that of branched polyethylenimine (25 kDa). In vitro gene transfection demonstrated that SP-PEI/DNA complex displayed increased transfection against 293T and SH-SY5Y cells with the increase of the weight ratio of SP-PEI/DNA complex with or without 10% serum. At weight ratio of 24, SP-PEI1800/DNA polyplexes showed the highest transfection on SH-SY5Y cells, which was almost three folds higher than PEI (25 kDa). Furthermore, these SP-PEIs/DNA polyplexes could effectively transfect 293T and SH-SY5Y cells with or without 10% serum, suggesting their excellent serum tolerance.

  17. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions....../translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models....

  18. Addition of Ascorbic Acid to the Extracellular Environment Activates Lipoplexes of a Ferrocenyl Lipid and Promotes Cell Transfection

    Science.gov (United States)

    Aytar, Burcu S.; Muller, John P. E.; Golan, Sharon; Hata, Shinichi; Takahashi, Hiro; Kondo, Yukishige; Talmon, Yeshayahu; Abbott, Nicholas L.; Lynn, David M.

    2011-01-01

    The level of cell transfection mediated by lipoplexes formed using the ferrocenyl lipid bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) depends strongly on the oxidation state of the two ferrocenyl groups of the lipid (reduced BFDMA generally mediates high levels of transfection, but oxidized BFDMA mediates very low levels of transfection). Here, we report that it is possible to chemically transform inactive lipoplexes (formed using oxidized BFMDA) to “active” lipoplexes that mediate high levels of transfection by treatment with the small-molecule reducing agent ascorbic acid (vitamin C). Our results demonstrate that this transformation can be conducted in cell culture media and in the presence of cells by addition of ascorbic acid to lipoplex-containing media in which cells are growing. Treatment of lipoplexes of oxidized BFDMA with ascorbic acid resulted in lipoplexes composed of reduced BFDMA, as characterized by UV/vis spectrophotometry, and lead to activated lipoplexes that mediated high levels of transgene expression in the COS-7, HEK 293T/17, HeLa, and NIH 3T3 cell lines. Characterization of internalization of DNA by confocal microscopy and measurements of the zeta potentials of lipoplexes suggested that these large differences in cell transfection result from (i) differences in the extents to which these lipoplexes are internalized by cells and (ii) changes in the oxidation state of BFDMA that occur in the extracellular environment (i.e., prior to internalization of lipoplexes by cells). Characterization of lipoplexes by small-angle neutron scattering (SANS) and by cryogenic transmission electron microscopy (cryo-TEM) revealed changes in the nanostructures of lipoplexes upon the addition of ascorbic acid, from aggregates that were generally amorphous, to aggregates with a more extensive multilamellar nanostructure. The results of this study provide guidance for the design of redox-active lipids that could lead to methods that enable spatial

  19. 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.

  20. 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

  1. Activity of levofloxacin alone and in combination with a DnaK inhibitor against gram-negative rods, including levofloxacin-resistant strains.

    Science.gov (United States)

    Credito, Kim; Lin, Gengrong; Koeth, Laura; Sturgess, Michael A; Appelbaum, Peter C

    2009-02-01

    Synergy time-kill testing of levofloxacin alone and in combination with CHP-105, a representative DnaK inhibitor, against 50 gram-negative rods demonstrated that 34 of the 50 strains tested showed significant synergy between levofloxacin and CHP-105 after 12 h and 24 h. Fourteen of these 34 organisms were quinolone resistant (levofloxacin MICs of > or =4 microg/ml).

  2. 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

  3. Activity of Levofloxacin Alone and in Combination with a DnaK Inhibitor against Gram-Negative Rods, Including Levofloxacin-Resistant Strains▿

    Science.gov (United States)

    Credito, Kim; Lin, Gengrong; Koeth, Laura; Sturgess, Michael A.; Appelbaum, Peter C.

    2009-01-01

    Synergy time-kill testing of levofloxacin alone and in combination with CHP-105, a representative DnaK inhibitor, against 50 gram-negative rods demonstrated that 34 of the 50 strains tested showed significant synergy between levofloxacin and CHP-105 after 12 h and 24 h. Fourteen of these 34 organisms were quinolone resistant (levofloxacin MICs of ≥4 μg/ml). PMID:19015359

  4. Islands of non-essential genes, including a DNA translocation operon, in the genome of bacteriophage 0305ϕ8-36

    Science.gov (United States)

    Pathria, Saurav; Rolando, Mandy; Lieman, Karen; Hayes, Shirley; Hardies, Stephen; Serwer, Philip

    2012-01-01

    We investigate genes of lytic, Bacillus thuringiensis bacteriophage 0305ϕ8-36 that are non-essential for laboratory propagation, but might have a function in the wild. We isolate deletion mutants to identify these genes. The non-permutation of the genome (218.948 Kb, with a 6.479 Kb terminal repeat and 247 identified orfs) simplifies isolation of deletion mutants. We find two islands of non-essential genes. The first island (3.01% of the genomic DNA) has an informatically identified DNA translocation operon. Deletion causes no detectable growth defect during propagation in a dilute agarose overlay. Identification of the DNA translocation operon begins with a DNA relaxase and continues with a translocase and membrane-binding anchor proteins. The relaxase is in a family, first identified here, with homologs in other bacteriophages. The second deleted island (3.71% of the genome) has genes for two metallo-protein chaperonins and two tRNAs. Deletion causes a significant growth defect. In addition, (1) we find by “in situ” (in-plaque) single-particle fluorescence microscopy that adsorption to the host occurs at the tip of the 486 nm long tail, (2) we develop a procedure of 0305ϕ8-36 purification that does not cause tail contraction, and (3) we then find by electron microscopy that 0305ϕ8-36 undergoes tail tip-tail tip dimerization that potentially blocks adsorption to host cells, presumably with effectiveness that increases as the bacteriophage particle concentration increases. These observations provide an explanation of the previous observation that 0305ϕ8-36 does not lyse liquid cultures, even though 0305ϕ8-36 is genomically lytic. PMID:22666654

  5. Diverse responses to UV light exposure in Acinetobacter include the capacity for DNA damage-induced mutagenesis in the opportunistic pathogens Acinetobacter baumannii and Acinetobacter ursingii

    Science.gov (United States)

    Bradley, James A.; Lin, Ching-li; Elam, Tyler J.

    2012-01-01

    Error-prone and error-free DNA damage repair responses that are induced in most bacteria after exposure to various chemicals, antibiotics or radiation sources were surveyed across the genus Acinetobacter. The error-prone SOS mutagenesis response occurs when DNA damage induces a cell’s umuDC- or dinP-encoded error-prone polymerases. The model strain Acinetobacter baylyi ADP1 possesses an unusual, regulatory umuD allele (umuDAb) with an extended 5′ region and only incomplete fragments of umuC. Diverse Acinetobacter species were investigated for the presence of umuDC and their ability to conduct UV-induced mutagenesis. Unlike ADP1, most Acinetobacter strains possessed multiple umuDC loci containing either umuDAb or a umuD allele resembling that of Escherichia coli. The nearly omnipresent umuDAb allele was the ancestral umuD in Acinetobacter, with horizontal gene transfer accounting for over half of the umuDC operons. Despite multiple umuD(Ab)C operons in many strains, only three species conducted UV-induced mutagenesis: Acinetobacter baumannii, Acinetobacter ursingii and Acinetobacter beijerinckii. The type of umuDC locus or mutagenesis phenotype a strain possessed was not correlated with its error-free response of survival after UV exposure, but similar diversity was apparent. The survival of 30 Acinetobacter strains after UV treatment ranged over five orders of magnitude, with the Acinetobacter calcoaceticus–A. baumannii (Acb) complex and haemolytic strains having lower survival than non-Acb or non-haemolytic strains. These observations demonstrate that a genus can possess a range of DNA damage response mechanisms, and suggest that DNA damage-induced mutation could be an important part of the evolution of the emerging pathogens A. baumannii and A. ursingii. PMID:22117008

  6. 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

  7. 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™.

  8. 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.

  9. 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.

  10. 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.

  11. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection

    NARCIS (Netherlands)

    Hujaya, S.D.; Marchioli, G.; Roelofs-Haarhuis, Hendrika Maria; van Apeldoorn, Aart A.; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Paulusse, Jos Marie Johannes; Engbersen, Johannes F.J.

    2015-01-01

    Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D.

  12. A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo

    Science.gov (United States)

    Hu, Jing; Zhu, Manman; Fan, Hua; Zhao, Wenfang; Mao, Yuan; Zhang, Yaguang

    2016-01-01

    Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100–300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo. PMID:27935984

  13. Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

    Science.gov (United States)

    Prezioso, Carla; Scribano, Daniela; Bellizzi, Anna; Anzivino, Elena; Rodio, Donatella Maria; Trancassini, Maria; Palamara, Anna Teresa; Pietropaolo, Valeria

    2017-12-01

    John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

  14. 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...

  15. 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.

  16. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions....... The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

  17. New water-soluble copper (II) complexes including 4,7-dimethyl-1,10-phenanthroline and L-tyrosine: Synthesis, characterization, DNA interactions and cytotoxicities

    Science.gov (United States)

    İnci, Duygu; Aydın, Rahmiye; Yılmaz, Dilek; Gençkal, Hasene Mutlu; Vatan, Özgür; Çinkılıç, Nilüfer; Zorlu, Yunus

    2015-02-01

    Two new water-soluble copper(II) complexes, [Cu(dmphen)2(NO3)]NO3 (1), [Cu(dmphen)(tyr)(H2O)]NO3·H2O (2) and the diquarternary salt of dmphen (dmphen = 4,7-dimethyl-1,10-phenanthroline and tyr = L-tyrosine), have been synthesized and characterized by elemental analysis, 1H NMR, 13C NMR and IR spectroscopy, thermal analysis and single crystal X-ray diffraction techniques. The CT-DNA binding properties of these compounds have been investigated by absorption, emission spectroscopy and thermal denaturation measurements. The supercoiled pBR322 plasmid DNA cleavage activity of these compounds has been explored by agarose gel electrophoresis. The cytotoxicity of these compounds against MCF-7, Caco-2, A549 cancer cells and BEAS-2B healthy cells was also studied by the XTT method. Complexes 1 and 2 exhibit significant cytotoxicity, with lower IC50 values than those of cisplatin.

  18. 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

  19. [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.

  20. 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 ...

  1. 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.

  2. 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.)

  3. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    Science.gov (United States)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  4. Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

    Directory of Open Access Journals (Sweden)

    Karina Hatamoto Kawasato

    2013-02-01

    Full Text Available Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP, the internal transcribed spacer 16S-23S rRNA (ITS and the cell division (FtsZ of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%, FtsZ (17.4% and ITS (21.7%, respectively. After the second round six positive samples were identified by nested-HSP (26%, eight by nested-ITS (34.8% and 18 by nested-FtsZ (78.2%, corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001, enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%. In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

  5. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    Science.gov (United States)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  6. 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.

  7. Mitochondrial DNA.

    Science.gov (United States)

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  8. Biophysical effects in off-resonant gold nanoparticle mediated (GNOME) laser transfection of cell lines, primary- and stem cells using fs laser pulses.

    Science.gov (United States)

    Schomaker, Markus; Killian, Doreen; Willenbrock, Saskia; Heinemann, Dag; Kalies, Stefan; Ngezahayo, Anaclet; Nolte, Ingo; Ripken, Tammo; Junghanß, Christian; Meyer, Heiko; Murua Escobar, Hugo; Heisterkamp, Alexander

    2015-08-01

    Gold nanoparticle mediated (GNOME) laser transfection is a powerful technique to deliver small biologically relevant molecules into cells. However, the transfection of larger and especially negatively charged DNA remains challenging. The efficiency for pDNA was 0.57% using parameter that does not influence the endo- and exogenous DNA. In order to gain a deeper understanding of the actual molecule uptake process, the uptake efficiency was determined using molecules of different sizes. It was evaluated that uncharged dextran molecules (2000 kDa) were delivered with an efficiency of 68%. The intracellular distribution of injected molecules was visualized and larger molecules were primary found in the cytoplasm. Patch clamp measurements suggested a permeabilization time up to 15 minutes. The uptake efficiency depended on the size and charge of the molecule to deliver as well as the cell size. A minor role for transfection plays the cell type since primary stem cells were successfully transfected. The perforation efficiency of semi-adherent and suspension cells is influenced by the cell and molecule size. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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

  10. 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

  11. 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.

  12. A new delimitation of the Afro-Eurasian plant genus Althenia to include its Australasian relative, Lepilaena (Potamogetonaceae) - Evidence from DNA and morphological data.

    Science.gov (United States)

    Ito, Yu; Tanaka, Norio; García-Murillo, Pablo; Muasya, A Muthama

    2016-05-01

    Althenia (Potamogetonaceae) is an aquatic plant genus disjunctly distributed in the southern- (South Africa's Cape Floristic Region: CFR) and northern- (Mediterranean Eurasia) hemispheres. This genus and its Australasian relative, Lepilaena, share similar floral characters yet have been treated as different genera or sections of Althenia sensu lato (s.l.) due to the isolated geographic distribution as well as the differences in sex expression, stamen construction, and stigma morphology. The diagnostic characters, however, need reevaluation over the boundaries between the entities. Here we tested the taxonomic delimitation between the entities, assessed synapomorphies for evolutionary lineages, and inferred biogeographic history in a phylogenetic framework. Our results indicated that Lepilaena was resolved as non-monophyletic in both plastid DNA and nuclear PhyC trees and Althenia was nested within it. As Althenia has nomenclatural priority, we propose a new delimitation to recognize Althenia s.l., which can be diagnosed by the female flowers with 3-segmented perianths and male flowers with perianths. The previously used diagnostic characters are either autapomorphies or synapomorphies for small lineages within Althenia s.l., and evolutionary transitions to sessile female flowers and narrow leaves characterize larger clades. Biogeographic analyses suggested a Miocene origin of Althenia s.l. in Australasia and indicated at least one inter- and one intra-specific inter-continental dispersal events among Australasia, Mediterranean Eurasia, and CFR need to be hypothesized to explain the current distribution patterns. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Preparation and characterization of polyethylenimine-coated Fe3O4-MCM-48 nanocomposite particles as a novel agent for magnet-assisted transfection.

    Science.gov (United States)

    Yiu, Humphrey H P; McBain, Stuart C; Lethbridge, Zoe A D; Lees, Martin R; Dobson, Jon

    2010-01-01

    A new type of magnetic nanoparticle was synthesized using mesoporous silica MCM-48 as a template. Magnetite (Fe(3)O(4)) nanocrystals were incorporated onto the MCM-48 silica structure by thermal decomposition of iron(III) acetylacetonate. The particle size of these Fe(3)O(4)-MCM-48 composite particles is around 300 nm with an iron oxide content of ca. 20% w/w. Measurements from SQUID magnetometry suggest that these nanoparticles possess superparamagnetic properties similar to those of Fe(3)O(4) nanoparticles. By coating positively charged polyethylenimine on to the surface, DNA can be bound onto the Fe(3)O(4)-MCM-48 nanoparticles. Transfection studies showed that these PEI-Fe(3)O(4)-MCM-48 particles were highly effective as a transfection reagent, and a 400% increase of transfection efficiency compared with the commercial products was recorded.

  14. 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.

  15. 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

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. 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.

  19. Using multi-scale molecular simulations to guide experimental design of biomaterials for drug and DNA delivery

    Science.gov (United States)

    Jayaraman, Arthi

    2015-03-01

    In this talk I will present molecular simulations studies that guide experimental synthesis of polymers for efficient DNA delivery. Viruses, while effective at delivery and transfection of DNA, can elicit harmful immunogenic responses, thus motivating design of non-viral transfection agents. Polycations are a promising class of non-viral vectors that bind to the negatively charged DNA backbone to form a complex (polyplex) that is then internalized into the target cell. Combinatorial approaches have generated various polycations with differing DNA transfection efficacies, but there is a need for general design guidelines that can relate the molecular features of the polycation to its DNA transfection efficiency. Using atomistic and coarse-grained molecular dynamics simulations we connect the thermodynamics of polycation-DNA binding and polyplex structure to experimentally observed transfection efficiency as a function of polycation chemistry and architecture.

  20. Electroporation-based DNA delivery technology

    DEFF Research Database (Denmark)

    Gothelf, A; Gehl, Julie

    2014-01-01

    DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses....

  1. 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.

  2. 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

  3. Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ahn Jae

    2010-05-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP and brain-derived neurotropfic factor (BDNF plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells. Results Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83% and only minimal cell damage than when conventional liposome-based reagent (in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells. Conclusion Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.

  4. Enhanced transfection of tumor cells in vivo using “Smart” pH-sensitive TAT-modified pegylated liposomes

    Science.gov (United States)

    Kale, Amit A.; Torchilin, Vladimir P.

    2012-01-01

    Liposomes have been prepared loaded with DNA (plasmid encoding for the green fluorescent protein, GFP) and additionally modified with TATp and PEG, with PEG being attached to the liposome surface via both pH-sensitive hydrazone and non-pH-sensitive bonds. The pGFP-loaded liposomal preparations have been administered intratumorarly in tumor-bearing mice and the efficacy of tumor cell transfection was followed after 72 h. The administration of pGFP–TATp–liposomes with non-pH-sensitive PEG coating has resulted in only minimal transfection of tumor cells because of steric hindrances for the liposome-to-cell interaction created by the PEG coat, which shielded the surface-attached TATp. At the same time, the administration of pGFP–TATp–liposomes with the low pH-detachable PEG resulted in at least three times more efficient transfection since the removal of PEG under the action of the decreased intratumoral pH leads to the exposure of the liposome-attached TATp residues, enhanced penetration of the liposomes inside tumor cells and more effective intracellular delivery of the pGFP. This result can be considered as an important step in the development of tumor-specific stimuli-sensitive drug and gene delivery systems. PMID:17671900

  5. 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.

  6. Use of damaged plasmid to study DNA repair in X-ray sensitive (xrs) strains of Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Smith-Ravin, J.; Jeggo, P.A.

    1989-01-01

    The effect of γ-irradiation of pSV2gpt DNA on its transfection frequency has been analysed using radiosensitive CHO xrs mutants showing a defect in double-strand break (dsb) rejoining. At low doses a sharp decrease in relative transfection frequency, i.e. transfection frequency of irradiated plasmid relative to untreated plasmid, as observed in xrs mutants compared with the parent line K1. Electrophoresis of irradiated plasmid DNA showed the decrease in transfection frequency in the xrs mutants correlated with the change of supercoiled molecules into open-circular forms. In the parent line CHO-K1, open-circular and supercoiled molecules have the same transfection frequency. The effect of linearization of pSV2gpt DNA by restriction enzymes on transfection frequency in xrs and wild-type strains was also examined. No difference in the relative transfection frequency between xrs and wild-type strains was detected. (author)

  7. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

  8. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

    Science.gov (United States)

    Steingrube, V A; Wilson, R W; Brown, B A; Jost, K C; Blacklock, Z; Gibson, J L; Wallace, R J

    1997-04-01

    A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

  9. Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts.

    Science.gov (United States)

    Kraja, Iv; Bing, Renjie; Hiwatashi, Nao; Rousseau, Bernard; Nalband, Danielle; Kirshenbaum, Kent; Branski, Ryan C

    2017-07-01

    An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent. In vitro. We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues. The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility. NA. Laryngoscope, 127:E231-E237, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  10. The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells.

    Science.gov (United States)

    Raof, Nurazhani A; Rajamani, Deepa; Chu, Hsun-Chieh; Gurav, Aniket; Johnson, Joel M; LoGerfo, Frank W; Pradhan-Nabzdyk, Leena; Bhasin, Manoj

    2016-01-05

    RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a "noncoding RNAi". However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA

  11. Comparison of chitosan, alginate and chitosan/alginate nanoparticles with respect to their size, stability, toxicity and transfection

    Directory of Open Access Journals (Sweden)

    Aras Rafiee

    2014-10-01

    Full Text Available Objective: To to compare the chitosan/alginate, chitosan and alginate nanoparticles as plasmid vectors, to determine the morphological characteristics, size and physicochemical properties of nanoparticle-pEGFP complexes and to evaluate the potential of these nanoparticles in transfection of pEGFP plasmid in to a cultured the human embryonic kidney cell line (HEK 293 cells. Methods: Nanoparticles comprising chitosan, alginate and both chitosan-alginate polymers were formed through pregel preparation method. The ability of plasmid-complexes in preventing DNA migration were assessed by the agarose gel assay. The efficiency of nanoparticles in transfection of pEGFP plasmid in the cultured HEK 293 cells was measured by flow cytometry. The effect of the nanoparticle-plasmid complexes on the cell viability was determined using cytotoxicity assay. Results: Chitosan, alginate and alginate/chitosan nanoparticles had a mean Z-average diameter of 620 nm, 235.8 nm and 161.8 nm and mean zeta potential of 45 mV, -18.6 mV and 29.3 mV, respectively. Chitosan and chitosan/alginate nanoparticles have greater capacity to maintain plasmid than alginate nanoparticles. Alginate nanoparticles had the greater transfection in comparison to the others. Cell viability assays indicated that nanoparticles had no toxic effect on HEK 293 cells after 4 h or 24 h. Conclusions: The combination of particle surface, hydrophobicity size and zeta potential can influence on transfection efficiency and the cellular uptake of the nanoparticles. Our suitable candidate for gene delivery would be alginate/chitosan nanoparticles.

  12. Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency.

    Science.gov (United States)

    Zhao, Xiubo; Pan, Fang; Zhang, ZhuoQi; Grant, Colin; Ma, YingHua; Armes, Steven P; Tang, YiQing; Lewis, Andrew L; Waigh, Thomas; Lu, Jian R

    2007-11-01

    Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.

  13. The effect of pegylation on the transfection activity of two ...

    African Journals Online (AJOL)

    Pegylated liposomes (80 - 150 nm diameter) formed electrostatic complexes with plasmid DNA in the 1.5:1 – 2.5:1 range of liposome (positive): DNA (negative) charge ratio, which afforded protection to the DNA cargo against serum nuclease digestion. Plasmid pGL3-containing pegylated lipoplexes were only weakly ...

  14. 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

  15. 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.

  16. 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

  17. 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....

  18. 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...

  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. Intelligent layered nanoflare: ``lab-on-a-nanoparticle'' for multiple DNA logic gate operations and efficient intracellular delivery

    Science.gov (United States)

    Yang, Bin; Zhang, Xiao-Bing; Kang, Li-Ping; Huang, Zhi-Mei; Shen, Guo-Li; Yu, Ru-Qin; Tan, Weihong

    2014-07-01

    DNA strand displacement cascades have been engineered to construct various fascinating DNA circuits. However, biological applications are limited by the insufficient cellular internalization of naked DNA structures, as well as the separated multicomponent feature. In this work, these problems are addressed by the development of a novel DNA nanodevice, termed intelligent layered nanoflare, which integrates DNA computing at the nanoscale, via the self-assembly of DNA flares on a single gold nanoparticle. As a ``lab-on-a-nanoparticle'', the intelligent layered nanoflare could be engineered to perform a variety of Boolean logic gate operations, including three basic logic gates, one three-input AND gate, and two complex logic operations, in a digital non-leaky way. In addition, the layered nanoflare can serve as a programmable strategy to sequentially tune the size of nanoparticles, as well as a new fingerprint spectrum technique for intelligent multiplex biosensing. More importantly, the nanoflare developed here can also act as a single entity for intracellular DNA logic gate delivery, without the need of commercial transfection agents or other auxiliary carriers. By incorporating DNA circuits on nanoparticles, the presented layered nanoflare will broaden the applications of DNA circuits in biological systems, and facilitate the development of DNA nanotechnology.DNA strand displacement cascades have been engineered to construct various fascinating DNA circuits. However, biological applications are limited by the insufficient cellular internalization of naked DNA structures, as well as the separated multicomponent feature. In this work, these problems are addressed by the development of a novel DNA nanodevice, termed intelligent layered nanoflare, which integrates DNA computing at the nanoscale, via the self-assembly of DNA flares on a single gold nanoparticle. As a ``lab-on-a-nanoparticle'', the intelligent layered nanoflare could be engineered to perform a variety of

  1. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  2. 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.

  3. Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K).

    Science.gov (United States)

    Singh, Isha; Kim, Myong-Jung; Molt, Robert W; Hoshika, Shuichi; Benner, Steven A; Georgiadis, Millie M

    2017-11-17

    A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host-guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs.

  4. 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.

  5. Neurotensin-Conjugated Reduced Graphene Oxide with Multi-Stage Near-Infrared-Triggered Synergic Targeted Neuron Gene Transfection In Vitro and In Vivo for Neurodegenerative Disease Therapy.

    Science.gov (United States)

    Hsieh, Tsung-Ying; Huang, Wei-Chen; Kang, Yi-Da; Chu, Chao-Yi; Liao, Wen-Lin; Chen, You-Yin; Chen, San-Yuan

    2016-12-01

    Delivery efficiency with gene transfection is a pivotal point in achieving maximized therapeutic efficacy and has been an important challenge with central nervous system (CNS) diseases. In this study, neurotensin (NT, a neuro-specific peptide)-conjugated polyethylenimine (PEI)-modified reduced graphene oxide (rGO) nanoparticles with precisely controlled two-stage near-infrared (NIR)-laser photothermal treatment to enhance the ability to target neurons and achieve high gene transfection in neurons. First-stage NIR laser irradiation on the cells with nanoparticles attached on the surface can increase the permeability of the cell membrane, resulting in an apparent increase in cellular uptake compared to untreated cells. In addition, second-stage NIR laser irradiation on the cells with nanoparticles inside can further induce endo/lysosomal cavitation, which not only helps nanoparticles escape from endo/lysosomes but also prevents plasmid DNA (pDNA) from being digested by DNase I. At least double pDNA amount can be released from rGO-PEI-NT/pDNA under NIR laser trigger release compared to natural release. Moreover, in vitro differentiated PC-12 cell and in vivo mice (C57BL/6) brain transfection experiments have demonstrated the highest transfection efficiency occurring when NT modification is combined with external multi-stage stimuli-responsive NIR laser treatment. The combination of neuro-specific targeting peptide and external NIR-laser-triggered aid provides a nanoplatform for gene therapy in CNS diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. 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

  7. Oral DNA Vaccine in Chickens

    Directory of Open Access Journals (Sweden)

    Seyed Davoud Jazayeri

    2012-01-01

    Full Text Available Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2% and MCF-10A (0.5% human breast cancer cells. Newly hatched specific-pathogen-free (SPF chicks were inoculated once by oral gavage with 109 colony-forming unit (CFU of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH and polymerase chain reaction (PCR were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

  8. 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.

  9. Construction of porcine CCK pDNA and its expression in COS-7 cells.

    Science.gov (United States)

    Bai, Jigang; Lü, Yi; Bai, Qiaoling

    2007-06-01

    CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamin 2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.

  10. A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes

    International Nuclear Information System (INIS)

    Jiang Yongjian; Jin Chen; Yang Feng; Yu Xianjun; Wang Guojian; Cheng Si; Di, Yang; Li, Ji; Fu, Deliang; N, Quanxing

    2011-01-01

    Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic interactions and shuttle the DNA to the cell cytoplasm or even the nucleus. Here we report a new approach to produce amino-CNTs by cycloaddition of nitrenes. Fourier transform infrared spectroscopy was used to verify the success of the functionalization, and the functionalization degree was calculated by thermal gravity analysis. Transmission electron microscope (TEM) was used to observe the solubility of the CNTs and the interactions of the amino-CNTs with the plasmids. Cell ultrathin sections were made and observed under the TEM to confirm the amino-CNTs enter the cells. Transfection experiments ultimately verify the amino-CNTs produced through cycloadditions of nitrenes can serve as plasmid vector.

  11. Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases.

    Science.gov (United States)

    Behzadi, Elham; Halabian, Raheleh; Hosseini, Hamideh Mahmoodzadeh; Fooladi, Abbas Ali Imani

    2016-11-01

    DNA vaccination -a third generation vaccine-is a modern approach to stimulate humoral and cellular responses against different diseases such as infectious diseases, cancer and autoimmunity. These vaccines are composed of a gene that encodes sequences of a desired protein under control of a proper (eukaryotic or viral) promoter. Immune response following DNA vaccination is influenced by the route and the dose of injection. In addition, antigen presentation following DNA administration has three different mechanisms including antigen presentation by transfected myocytes, transfection of professional antigen presenting cells (APCs) and cross priming. Recently, it has been shown that bacterial toxins and their components can stimulate and enhance immune responses in experimental models. A study demonstrated that DNA fusion vaccine encoding the first domain (DOM) of the Fragment C (FrC) of tetanus neurotoxin (CTN) coupled with tumor antigen sequences is highly immunogenic against colon carcinoma. DNA toxin vaccines against infectious and autoimmune diseases are less studied until now. All in all, this novel approach has shown encouraging results in animal models, but it has to go through adequate clinical trials to ensure its effectiveness in human. However, it has been proven that these vaccines are safe, multifaceted and simple and can be used widely in organisms which may be of advantage to public health in the near future. This paper outlines the mechanism of the action of DNA vaccines and their possible application for targeting infectious diseases, cancer and autoimmunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. 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.

  13. Polyplex micelles with thermoresponsive heterogeneous coronas for prolonged blood retention and promoted gene transfection.

    Science.gov (United States)

    Li, Yang; Li, Junjie; Chen, Biao; Chen, Qixian; Zhang, Guoying; Liu, Shiyong; Ge, Zhishen

    2014-08-11

    Adequate retention in blood circulation is a prerequisite for construction of gene delivery carriers for systemic applications. The stability of gene carriers in the bloodstream requires them to effectively resist protein adsorption and maintain small size in the bloodstream avoiding dissociation, aggregation, and nuclease digestion under salty and proteinous medium. Herein, a mixture of two block catiomers consisting of the same cationic block, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), but varying shell-forming blocks, poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEO2MA), and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), was used to complex with plasmid DNA (pDNA) to fabricate polyplex micelles with mixed shells (MPMs) at 20 °C. The thermoresponsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 to 37 °C, which results in a distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of the MPMs. Subsequent study verified that this transition promoted further condensation of pDNA, thereby giving rise to improved complex and colloidal stability. The proposed system has shown remarkable stability in salty and proteinous solution and superior tolerance to nuclease degradation. As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET) block copolymer, in vivo circulation experiments in the bloodstream further confirmed that the retention time of MPMs was prolonged significantly. Moreover, the proposed system exhibited remarkably high cell transfection activity especially at low N/P ratios and negligible cytotoxicity and thus portends promising utility for systemic gene therapy applications.

  14. 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.

  15. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  16. Transfection of a retroviral construct carrying a non producer HIV-1 variant induces HIV-1 resistance in CD4+ CEMss cells.

    Science.gov (United States)

    Federico, M; Taddeo, B; Nappi, F; Nicolini, A; Rossi, G B; Verani, P

    1993-01-01

    The phenotype of Human Immunodeficiency Virus-1 (HIV)-infected HUT-78 cell clone (F12) has been described (Federico et al, AIDS Res Hum Retrov 1989; 5: 365-96). Briefly, F12 cells are: i) CD4 down-regulated, ii) non producer and iii) fully resistant to homologous superinfection. We tested whether this phenotype was dependent upon the expression of the HIV-1 genome integrated therein. The SstI/SstI F12 provirus was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo)-Geneticine resistance gene. CD4+ HIV-susceptible CEMss cells were transfected with this construct in the sense orientation. Neo-resistant clones exhibited an integrated viral DNA, low viral mRNA expression and (as in F12 cells) the presence of uncleaved gp160, no gp41 and a small amount of p55 gag precursor. Superinfection of the F12/HIV-DNA-transfected CEMss clones showed that these CD4+ cells had acquired a significant (0.7-1.5 logs) resistance towards superinfection with HIV-1. This was observed in all four transfected clones where the F12/HIV DNA was expressed, but not in the control clone that was transfected with the pLj vector alone. These results confirm those that were obtained with human CD4+ CEMss cells infected with a recombinant retrovirus bearing the same SstI/SstI F12/HIV genome (Federico et al, J Gen Virol, 1993, in press). Both sets of results indicate that the expression of this genome in bio-engineered CD4+ human cells results in their intracellular immunization against HIV-1.

  17. Nucleic Acid Sensors Involved in the Recognition of HBV in the Liver–Specific in vivo Transfection Mouse Models—Pattern Recognition Receptors and Sensors for HBV

    Directory of Open Access Journals (Sweden)

    Chean Ring Leong

    2015-04-01

    Full Text Available Cellular innate immune system recognizing pathogen infection is critical for the host defense against viruses. Hepatitis B virus (HBV is a DNA virus with a unique life cycle whereby the DNA and RNA intermediates present at different phases. However, it is still unclear whether the viral DNA or RNA templates are recognized by the pattern-recognition receptors (PRRs to trigger host antiviral immune response. Here in this article, we review the recent advances in the progress of the HBV studies, focusing on the nucleic acid sensors and the pathways involved in the recognition of HBV in the liver–specific in vivo transfection mouse models. Hydrodynamic injection transfecting the hepatocytes in the gene-disrupted mouse model with the HBV replicative genome DNA has revealed that IFNAR and IRF3/7 are indispensable in HBV eradication in the mice liver but not the RNA sensing pathways. Interestingly, accumulating evidence of the recent studies has demonstrated that HBV markedly interfered with IFN-β induction and antiviral immunity mediated by the Stimulator of interferon genes (STING, which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. This review will present the current understanding of innate immunity in HBV infection and of the challenges for clearing of the HBV infection.

  18. Fabrication and characterization of DNA-loaded zein nanospheres

    Directory of Open Access Journals (Sweden)

    Regier Mary C

    2012-12-01

    Full Text Available Abstract Background Particulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles. Results Zein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization. Conclusions This study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated

  19. Fabrication and characterization of DNA-loaded zein nanospheres.

    Science.gov (United States)

    Regier, Mary C; Taylor, Jessica D; Borcyk, Tyler; Yang, Yiqi; Pannier, Angela K

    2012-12-02

    Particulates incorporating DNA are promising vehicles for gene delivery, with the ability to protect DNA and provide for controlled, localized, and sustained release and transfection. Zein, a hydrophobic protein from corn, is biocompatible and has properties that make it a promising candidate material for particulate delivery, including its ability to form nanospheres through coacervation and its insolubility under physiological conditions, making it capable of sustained release of encapsulated compounds. Due to the promise of this natural biomaterial for drug delivery, the objective of this study was to formulate zein nanospheres encapsulating DNA as the therapeutic compound, and to characterize size, charge, sustained release, cell cytotoxicity and cellular internalization of these particles. Zein nanospheres encapsulating DNA were fabricated using a coacervation technique, without the use of harsh solvents or temperatures, resulting in the preservation of DNA integrity and particles with diameters that ranged from 157.8 ± 3.9 nm to 396.8 ± 16.1 nm, depending on zein to DNA ratio. DNA encapsulation efficiencies were maximized to 65.3 ± 1.9% with a maximum loading of 6.1 ± 0.2 mg DNA/g zein. The spheres protected encapsulated DNA from DNase I degradation and exhibited sustained plasmid release for at least 7 days, with minimal burst during the initial phase of release. Zein/DNA nanospheres demonstrated robust biocompatibility, cellular association, and internalization. This study represents the first report on the formation of zein particles encapsulating plasmid DNA, using simple fabrication techniques resulting in preservation of plasmid integrity and tunable sizes. DNA encapsulation efficiencies were maximized to acceptable levels at higher zein to DNA ratios, while loading was comparable to that of other hydrophilic compounds encapsulated in zein and that of DNA incorporated into PLGA nano- and microspheres. The hydrophobic nature of zein resulted in

  20. 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

  1. 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.

  2. 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.

  3. Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition

    Science.gov (United States)

    Pozzi, D.; Marchini, C.; Cardarelli, F.; Salomone, F.; Coppola, S.; Montani, M.; Zabaleta, M. Elexpuru; Digman, M.A.; Gratton, E.; Colapicchioni, V.; Caracciolo, G.

    2014-01-01

    Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid–protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP–DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP–DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol–DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. PMID:24296066

  4. The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain.

    Science.gov (United States)

    Ojeda, E; Puras, G; Agirre, M; Zarate, J; Grijalvo, S; Eritja, R; Martinez-Navarrete, G; Soto-Sánchez, C; Diaz-Tahoces, A; Aviles-Trigueros, M; Fernández, E; Pedraz, J L

    2016-01-01

    The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Chondrogenic effect of precartilaginous stem cells following NLS-TAT cell penetrating peptide-assisted transfection of eukaryotic hTGFβ3.

    Science.gov (United States)

    Guo, Xin; Chu, Xiangyu; Li, Wenkai; Pan, Qiyong; You, Hongbo

    2013-11-01

    Cell penetrating peptides (CPPs) are a series of promising carriers for delivering exogenous DNA to living cells. Among them, the combination of the human immunodeficiency virus TAT protein (TAT) with the SV40 large T protein nuclear localization signal (NLS) to form NLS-TAT performs well. In the present study, we took advantage of this new carrier to deliver transforming growth factor-beta 3 (TGFβ3) genes. TGFβ3 was expressed by the pEGFP-N1 vector following transfection of rat precartilaginous stem cells (PSCs), which promoted hTGFβ3 protein self-expression. At 24, 48, 72, and 120 h after transfection, the expression levels of hTGFβ3 were found to be elevated as compared with the control. The expression of hTGFβ3 was found to mediate the chondrogenic effect of PSCs. Thus, we determined the expression of the chondrogenesis-related genes type II collagen, Sox 9, and aggrecan in PSCs at 24, 48, 72, and 120 h after transfection. We found that their transcription and translation was augmented, which indicated a trend of active chondrogenesis in the PSCs. Our results demonstrated that NLS-TAT had the ability to deliver exogenous DNA into rat PSCs and could be actively expressed. This process successfully promoted PSC chondrogenesis. Additionally, PSC, may represent a new type of stem cells, and thus show great potential in regenerative repair following cartilage injury. © 2013 Wiley Periodicals, Inc.

  6. Student award winner in the Ph.D. category for the 2013 society for biomaterials annual meeting and exposition, april 10-13, 2013, Boston, Massachusetts : biomaterial-mediated cancer-specific DNA delivery to liver cell cultures using synthetic poly(beta-amino ester)s.

    Science.gov (United States)

    Tzeng, Stephany Y; Higgins, Luke J; Pomper, Martin G; Green, Jordan J

    2013-07-01

    Liver cancer is a leading cause of cancer death. Most patients are treated by arterial injection of chemoembolizing agents, providing a convenient avenue for local treatment by novel therapies, including gene therapy. Poly(beta-amino ester)s (PBAEs) were synthesized and used to form nanoparticles for non-viral transfection of buffalo rat hepatoma (MCA-RH7777) and hepatocyte (BRL-3A) lines with eGFP and luciferase DNA. Hepatoma cells were transfected with up to (98 ± 0.4)% efficacy with no measurable cytotoxicity. Hepatocytes were transfected with as high as (73 ± 0.4)% efficacy with (10 ± 4)% non-specific cytotoxicity. In contrast, positive controls (branched polyethylenimine, Lipofectamine™ 2000, and X-tremeGENE(®) DNA HP) caused 30-90% toxicity in BRL-3A cells at doses required for >50% transfection. Of the 21 optimized PBAE-DNA formulations tested, 12 showed significant specificity for hepatoma cells over hepatocytes in monoculture (p biomaterial-mediated specificity to prevent toxic side-effects on healthy hepatocytes. Copyright © 2013 Wiley Periodicals, Inc.

  7. Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds

    Czech Academy of Sciences Publication Activity Database

    Petráková, Vladimíra; Benson, Veronika; Bunček, M.; Fišerová, Anna; Ledvina, Miroslav; Štursa, Jan; Cígler, Petr; Nesládek, M.

    2016-01-01

    Roč. 8, č. 23 (2016), s. 12002-12012 ISSN 2040-3364 R&D Projects: GA MZd(CZ) NV15-33094A; GA MŠk LM2015056 Grant - others:OP VK(XE) CZ.1.07/2.3.00/20.0306 Institutional support: RVO:68378271 ; RVO:61388971 ; RVO:61389005 ; RVO:61388963 Keywords : single-particle tracking * resonance energy-transfer * sirna delivery * gene delivery * correlation spectroscopy * endosomal escape * mass-production * drug-delivery * quantum dots * living cells Subject RIV: BM - Solid Matter Physics ; Magnetism; CC - Organic Chemistry (UOCHB-X); EE - Microbiology, Virology (MBU-M); BG - Nuclear, Atomic and Molecular Physics, Colliders (UJF-V) Impact factor: 7.367, year: 2016

  8. The effects of MicroRNA transfections on global patterns of gene expression in ovarian cancer cells are functionally coordinated

    Directory of Open Access Journals (Sweden)

    Shahab Shubin W

    2012-08-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a class of small RNAs that have been linked to a number of diseases including cancer. The potential application of miRNAs in the diagnostics and therapeutics of ovarian and other cancers is an area of intense interest. A current challenge is the inability to accurately predict the functional consequences of exogenous modulations in the levels of potentially therapeutic miRNAs. Methods In an initial effort to systematically address this issue, we conducted miRNA transfection experiments using two miRNAs (miR-7, miR-128. We monitored the consequent changes in global patterns of gene expression by microarray and quantitative (real-time polymerase chain reaction. Network analysis of the expression data was used to predict the consequence of each transfection on cellular function and these predictions were experimentally tested. Results While ~20% of the changes in expression patterns of hundreds to thousands of genes could be attributed to direct miRNA-mRNA interactions, the majority of the changes are indirect, involving the downstream consequences of miRNA-mediated changes in regulatory gene expression. The changes in gene expression induced by individual miRNAs are functionally coordinated but distinct between the two miRNAs. MiR-7 transfection into ovarian cancer cells induces changes in cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions (EMT and other processes linked with metastasis. In contrast, miR-128 transfection induces changes in cell cycle control and other processes commonly linked with cellular replication. Conclusions The functionally coordinated patterns of gene expression displayed by different families of miRNAs have the potential to provide clinicians with a strategy to treat cancers from a systems rather than a single gene perspective.

  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. 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.

  11. Biodegradable cationic poly(carbonates): Effect of varying side chain hydrophobicity on key aspects of gene transfection.

    Science.gov (United States)

    Ong, Zhan Yuin; Yang, Chuan; Cheng, Wei; Voo, Zhi Xiang; Chin, Willy; Hedrick, James L; Yang, Yi Yan

    2017-05-01

    The degree of hydrophobicity in cationic polymers plays an important but often underappreciated role in the safety and efficacy of gene delivery processes. In order to further elucidate structure-activity relationships of biodegradable cationic poly(carbonate) gene carriers, we synthesized a series of narrowly dispersed homo-polymers via metal-free organocatalytic living ring-opening polymerization (ROP) of cyclic carbonate monomers bearing either alkyl (propyl, hexyl or nonyl) or 4-methyl benzyl halide side chains. The polymers were then quaternized using bis-tertiary amines to install both quaternary ammoniums and tertiary amines for DNA binding and endosomal escape, respectively. Among the polymers with similar molecular lengths and charge densities, it was found that an increase in side chain alkyl spacer length from 3 to 6 carbons significantly enhanced cellular uptake and luciferase gene expression in HepG2 and HeLa cell lines without causing overt hemolysis and cytotoxicity. A further increase of side chain alkyl length to 9 carbons, however, led to a drastic decline in gene expression due to increased cellular toxicity, which was correlated with an increased disruption and lysis of red blood cell membranes. Interestingly, the incorporation of an aromatic 4-methyl benzyl spacer increased DNA binding strength, reduced particle sizes of resultant DNA complexes, and enhanced cellular uptake, leading to improved luciferase gene expression, albeit with higher levels of hemolysis and cytotoxicity. Taken together, the findings of this study demonstrate that a delicate balance between cationic charge density and hydrophobicity could be achieved by utilizing a hexyl spacer in the side chains of cationic poly(carbonates), hence providing insights on the future development of non-viral cationic polymeric gene delivery systems. Owing to their ease of synthesis and well-controlled polymerization, biodegradable cationic poly(carbonates) have emerged as a highly promising

  12. Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhao QQ

    2012-06-01

    Full Text Available Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence was successfully synthesized and linked with chitosan-linked polyethylenimine (CP to form a new targeted gene delivery vector called CPT (CP/peptide. The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT

  13. 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.

  14. 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...

  15. 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 ...

  16. 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...

  17. [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.

  18. Mesoporous Silica Nanomaterials for Applications in Catalysis, Sensing, Drug Delivery and Gene Transfection

    Energy Technology Data Exchange (ETDEWEB)

    Radu, Daniela Rodica [Iowa State Univ., Ames, IA (United States)

    2004-01-01

    the surface of MSN and utilize them to complex cationic DNA. The p-EGFP-CI gene-coated MSN nanocomposite was able to transfect cancer cell lines, such as human HeLa and CHO cancer cell lines. The gene carrier ability of MSNs was further proved by transfecting primary cells and cotransfecting of two different genes in cancer cell lines. In sum, MSN are versatile partners in several types of applications.

  19. Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation.

    Science.gov (United States)

    Glahder, Jacob; Norrild, Bodil; Persson, Mikael B; Persson, Bertil R R

    2005-11-05

    Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1

  20. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  1. 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...

  2. Mixtures of cationic lipid O-ethylphosphatidylcholine with membrane lipids and DNA: phase diagrams.

    Science.gov (United States)

    Koynova, Rumiana; MacDonald, Robert C

    2003-10-01

    Ethylphosphatidylcholines are positively charged membrane lipid derivatives, which effectively transfect DNA into cells and are metabolized by the cells. For this reason, they are promising nonviral transfection agents. With the aim of revealing the kinds of lipid phases that may arise when lipoplexes interact with cellular lipids during DNA transfection, temperature-composition phase diagrams of mixtures of the O-ethyldipalmitoylphosphatidylcholine with representatives of the major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cholesterol) were constructed. Phase boundaries were determined using differential scanning calorimetry and synchrotron x-ray diffraction. The effects of ionic strength and of DNA presence were examined. A large variety of polymorphic and mesomorphic structures were observed. Surprisingly, marked enhancement of the affinity for nonlamellar phases was observed in mixtures with phosphatidylethanolamine and cholesterol as well as with phosphatidylglycerol (previously reported). Because of the potential relevance to transfection, it is noteworthy that such phases form at close to physiological conditions, and in the presence of DNA. All four mixtures exhibit a tendency to molecular clustering in the gel phase, presumably due to the specific interdigitated molecular arrangement of the O-ethyldipalmitoylphosphatidylcholine gel bilayers. It is evident that a remarkably broad array of lipid phases could arise in transfected cells and that these could have significant effects on transfection efficiency. The data may be particularly useful for selecting possible "helper" lipids in the lipoplex formulations, and in searches for correlations between lipoplex structure and transfection activity.

  3. Downregulation of NF-κB activation in a H4IIE transfectant insensitive to doxorubicin-induced apoptosis

    International Nuclear Information System (INIS)

    Chovolou, Yvonni; Waetjen, Wim; Kampkoetter, Andreas; Kahl, Regine

    2007-01-01

    Cytostatic drugs are administered to cancer patients in order to drive the tumor cells into apoptosis by DNA damage signalling pathway(s). DNA damage also leads to NF-κB activation, and it is controversial whether this is exclusively part of a survival process, thus enabling drug resistance, or whether it can also lead to a pro-apoptotic response, thus supporting the therapeutic purpose of drug administration. In the present work, the pathway and outcome of NF-κB activation was compared in the doxorubicin sensitive H4IIE rat hepatoma cell and the H4IIE-derived transfectant Yv2-12 which is insensitive to doxorubicin induced apoptosis. In the wild type H4IIE cell, doxorubicin induces serine 536 phosphorylation and nuclear translocation of p65 which however results in reduced rather than increased expression of the anti-apoptotic protein XIAP. Apoptosis in H4IIE cells is accompanied by rapid production of intracellular reactive oxygen species, caspase activation and increased expression of the pro-apoptotic protein Bax. The doxorubicin-insensitive Yv2-12 transfectant differs from its wild type counterpart by the complete failure to activate NF-κB in response to doxorubicin. In contrast, serine 536 phosphorylation and nuclear translocation of p65 are even reduced by doxorubicin treatment while the expression of XIAP and Bax remain virtually unchanged. These results show that NF-κB activation by doxorubicin in our experimental system proceeds by an atypical pathway resulting in a pro-apoptotic effect and that insensitivity to doxorubicin-induced apoptosis was accompanied by a loss of NF-κB activation

  4. 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.

  5. 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

  6. 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.

  7. 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)

  8. 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.

  9. Silicon supported lipid-DNA thin film structures at varying temperature studied by energy dispersive X-ray diffraction and neutron reflectivity.

    Science.gov (United States)

    Domenici, F; Castellano, C; Dell'Unto, F; Albinati, A; Congiu, A

    2011-11-01

    Non-viral gene transfection by means of lipid-based nanosystems, such as solid supported lipid assemblies, is often limited due to their lack of stability and the consequent loss of efficiency. Therefore not only a detailed thermo-lyotropic study of these DNA-lipid complexes is necessary to understand their interaction mechanisms, but it can also be considered as a first step in conceiving and developing new transfection biosystems. The aim of our study is a structural characterization of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-dimethyl-dioctadecyl-ammonium bromide (DDAB)-DNA complex at varying temperature using the energy dispersive X-ray diffraction (EDXD) and neutron reflectivity (NR) techniques. We have shown the formation of a novel thermo-lyotropic structure of DOPC/DDAB thin film self-organized in multi-lamellar planes on (100)-oriented silicon support by spin coating, thus enlightening its ability to include DNA strands. Our NR measurements indicate that the DOPC/DDAB/DNA complex forms temperature-dependent structures. At 65°C and relative humidity of 100% DNA fragments are buried between single lamellar leaflets constituting the hydrocarbon core of the lipid bilayers. This finding supports the consistency of the hydrophobic interaction model, which implies that the coupling between lipid tails and hypo-hydrated DNA single strands could be the driving force of DNA-lipid complexation. Upon cooling to 25°C, EDXD analysis points out that full-hydrated DOPC-DDAB-DNA can switch in a different metastable complex supposed to be driven by lipid heads-DNA electrostatic interaction. Thermotropic response analysis also clarifies that DOPC has a pivotal role in promoting the formation of our observed thermophylic silicon supported lipids-DNA assembly. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  11. 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...

  12. [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

  13. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  14. A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

    Science.gov (United States)

    Zhao, Deyao; Huang, Dong; Li, Yang; Wu, Mengxi; Zhong, Wenfeng; Cheng, Qiang; Wang, Xiaoxia; Wu, Yidi; Zhou, Xiao; Wei, Zewen; Li, Zhihong; Liang, Zicai

    2016-01-01

    Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.

  15. Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

    Science.gov (United States)

    Hashimoto, Tomoko; Kawazu, Takeshi; Nagasaki, Takeshi; Murakami, Akira; Yamaoka, Tetsuji

    2012-02-01

    Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

  16. 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.

  17. Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine.

    Science.gov (United States)

    Nematollahi, Mohammad Hadi; Torkzadeh-Mahanai, Masoud; Pardakhty, Abbas; Ebrahimi Meimand, Hossein Ali; Asadikaram, Gholamreza

    2017-10-28

    Non-viral gene delivery methods are considered due to safety and simplicity in human gene therapy. Since the use of cationic peptide and niosome represent a promising approach for gene delivery purposes we used recombinant fusion protein and cationic niosome as a gene carrier. A multi-domain fusion protein including nuclear localization motif (NLS) and two DNA-binding (Mu) domains, namely NLS-Mu-Mu (NMM) has been designed, cloned and expressed in E. coli DE3 strain. Afterward, the interested protein was purified by affinity chromatography. Binary vectors based on protein/DNA and ternary vectors based on protein/DNA/niosome were prepared. Protamine was used as a control. DNA condensing properties of NMM and protamine were evaluated by various experiments. Furthermore, we examined cytotoxicity, hemolysis and transfection potential of the binary and ternary complexes in HEK293T and MCF-7 cell lines. Protamine and Lipofectamine™2000 were used as positive controls, correspondingly. The recombinant NMM was expressed and purified successfully and DNA was condensed efficiently at charge ratios that were not harmful to cells. Peptidoplexes showed transfection efficiency (TE) but ternary complexes had higher TE. Additionally, NMM ternary complex was more efficient compared to protamine ternary vectors. Our results showed that niosomal ternary vector of NMM is a promising non-viral gene carrier to achieve an effective and safe carrier system for gene therapy.

  18. 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...

  19. 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...

  20. 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.

  1. Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

    Science.gov (United States)

    Park, Chan-Hwan; Ju, Hye-Kyoung; Han, Jae-Yeong; Park, Jong-Seo; Kim, Ik-Hyun; Seo, Eun-Young; Kim, Jung-Kyu; Hammond, John; Lim, Hyoun-Sub

    2017-04-01

    Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

  2. Development of DNA vaccines for fish

    DEFF Research Database (Denmark)

    Heppell, Joël; Lorenzen, Niels; Armstrong, Neil K.

    1998-01-01

    Disease control is one of the major concerns in the aquaculture industry. However, there are no vaccines available for the prevention of many piscine infectious diseases, especially those of viral and parasitic origin. DNA-based vaccination could circumvent several problems associated...... no permanent tissue damage. To further investigate the ability of DNA-based vaccines to induce protective immunity in fish, viral haemorrhagic septicaemia virus G and N genes were cloned individually into an expression plasmid. Both G and N proteins produced in transfected fish cells appeared identical...... protein, killing the transfected host cells and ablating further expression of G protein and luciferase. Finally, young rainbow trout injected with the G construct, alone or together with the N construct, were strongly protected against challenge with live virus. These results suggest that DNA vaccines...

  3. 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.

  4. [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.

  5. 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...

  6. Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

    Directory of Open Access Journals (Sweden)

    Jinghua Duan

    2009-01-01

    Full Text Available To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA and chitosan to prepare PBCA nanoparticles (NPs by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2 cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1 was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS. Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

  7. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  8. Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

    Science.gov (United States)

    Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

    1993-01-01

    We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

  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. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Phosphorylation of PTEN at STT motif is associated with DNA damage response

    International Nuclear Information System (INIS)

    Misra, Sandip; Mukherjee, Ananda; Karmakar, Parimal

    2014-01-01

    Highlights: • Phosphorylation PTEN at the C-terminal STT motif is necessary for DNA repair. • DNA damage induces phosphorylation of STT motif of PTEN. • Phospho-PTEN translocates to nucleus after DNA damage. • Phospho-PTEN forms nuclear foci after DNA damage which co localized with γH2AX. - Abstract: Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair

  16. Phosphorylation of PTEN at STT motif is associated with DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Sandip; Mukherjee, Ananda; Karmakar, Parimal, E-mail: pkarmakar_28@yahoo.co.in

    2014-12-15

    Highlights: • Phosphorylation PTEN at the C-terminal STT motif is necessary for DNA repair. • DNA damage induces phosphorylation of STT motif of PTEN. • Phospho-PTEN translocates to nucleus after DNA damage. • Phospho-PTEN forms nuclear foci after DNA damage which co localized with γH2AX. - Abstract: Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair.

  17. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

    Science.gov (United States)

    Gallego-Perez, Daniel; Pal, Durba; Ghatak, Subhadip; Malkoc, Veysi; Higuita-Castro, Natalia; Gnyawali, Surya; Chang, Lingqian; Liao, Wei-Ching; Shi, Junfeng; Sinha, Mithun; Singh, Kanhaiya; Steen, Erin; Sunyecz, Alec; Stewart, Richard; Moore, Jordan; Ziebro, Thomas; Northcutt, Robert G.; Homsy, Michael; Bertani, Paul; Lu, Wu; Roy, Sashwati; Khanna, Savita; Rink, Cameron; Sundaresan, Vishnu Baba; Otero, Jose J.; Lee, L. James; Sen, Chandan K.

    2017-10-01

    Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

  18. A cell spot microarray method for production of high density siRNA transfection microarrays

    Directory of Open Access Journals (Sweden)

    Mpindi John-Patrick

    2011-03-01

    Full Text Available Abstract Background High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. Results Here, we describe the optimization of a miniaturized cell spot microarray (CSMA method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. Conclusions The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.

  19. DNA fingerprinting of the NCI-60 cell line panel.

    Science.gov (United States)

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  20. Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

    OpenAIRE

    Zanta, Maria Antonietta; Belguise-Valladier, Pascale; Behr, Jean-Paul

    1999-01-01

    Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of ...

  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. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

    1988-01-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  3. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Energy Technology Data Exchange (ETDEWEB)

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  4. Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers.

    Science.gov (United States)

    Mahajan, Vivek; Gaymalov, Zagit; Alakhova, Daria; Gupta, Richa; Zucker, Irving H; Kabanov, Alexander V

    2016-01-01

    Intramuscular administration of plasmid DNA (pDNA) with non-ionic Pluronic block copolymers increases gene expression in injected muscles and lymphoid organs. We studied the role of immune cells in muscle transfection upon inflammation. Local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and expressed protein levels in ischemic muscles injected with pDNA/Pluronic. The systemic inflammation (MHLIM or peritonitis) also increased expression of pDNA/Pluronic in the muscles. When pDNA/Pluronic was injected in ischemic muscles the reporter gene, Green Fluorescent Protein (GFP) co-localized with desmin(+) muscle fibers and CD11b(+) macrophages (MØs), suggesting transfection of MØs along with the muscle cells. P85 enhanced (∼ 4 orders) transfection of MØs with pDNA in vitro. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells in MHLIM. Using a co-culture of myotubes (MTs) and transfected MØs expressing a reporter gene under constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) promoter we demonstrated that P85 enhances horizontal gene transfer from MØ to MTs. Therefore, MØs can play an important role in muscle transfection with pDNA/Pluronic during inflammation, with both inflammation and Pluronic contributing to the increased gene expression. pDNA/Pluronic has potential for therapeutic gene delivery in muscle pathologies that involve inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.

    Science.gov (United States)

    Le Bivic, A; Sambuy, Y; Patzak, A; Patil, N; Chao, M; Rodriguez-Boulan, E

    1991-11-01

    A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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

  8. Antinociceptive effects of morphine and naloxone in mu-opioid receptor knockout mice transfected with the MORS196A gene

    Directory of Open Access Journals (Sweden)

    Tao Pao-Luh

    2010-04-01

    Full Text Available Abstract Background Opioid analgesics such as morphine and meperidine have been used to control moderate to severe pain for many years. However, these opioids have many side effects, including the development of tolerance and dependence after long-term use, which has limited their clinical use. We previously reported that mutations in the mu-opioid receptors (MOR S196L and S196A rendered them responsive to the opioid antagonist naloxone without altering the agonist phenotype. In MORS196A knock-in mice, naloxone and naltrexone were antinociceptive but did not cause tolerance or physical dependence. In this study we delivery this mutated MOR gene into pain related pathway to confirm the possibility of in vivo transfecting MORS196A gene and using naloxone as a new analgesic agent. Methods The MOR-knockout (MOR-KO mice were used to investigate whether morphine and naloxone could show antinociceptive effects when MORS196A gene was transfected into the spinal cords of MOR-KO mice. Double-stranded adeno-associated virus type 2 (dsAAV2 was used to deliver the MORS196A-enhanced green fluorescence protein (EGFP gene by microinjected the virus into the spinal cord (S2/S3 dorsal horn region. Tail-flick test was used to measure the antinociceptive effect of drugs. Results Morphine (10 mg/kg, s.c. and naloxone (10 mg/kg, s.c. had no antinociceptive effects in MOR-KO mice before gene transfection. However, two or three weeks after the MOR-S196A gene had been injected locally into the spinal cord of MOR-KO mice, significant antinociceptive effects could be induced by naloxone or morphine. On the other hand, only morphine but not naloxone induced significant tolerance after sub-chronic treatment. Conclusion Transfecting the MORS196A gene into the spinal cord and systemically administering naloxone in MOR-KO mice activated the exogenously delivered mutant MOR and provided antinociceptive effect without causing tolerance. Since naloxone will not activate natural

  9. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  10. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    International Nuclear Information System (INIS)

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT + colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references

  11. 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

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

    Directory of Open Access Journals (Sweden)

    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

  13. 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

  14. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  15. 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.

  16. 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.

  17. [Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

    Science.gov (United States)

    Yu, Wen-juan; Wang, Yue-wei; Xie, Zhi-gang; You, Jiang-feng; Wang, Jie-liang; Cui, Xiang-lin; Pei, Fei; Zheng, Jie

    2010-02-01

    To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells. A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. DNA Translocation and Cell Electroporation in Micro and Nanofluidic Devices

    Science.gov (United States)

    Gupta, Cherry

    The cell membrane is made of a thin (˜ 5nm) lipid bilayer which serves as an effective insulator and diffusion barrier for entities external to the cell from entering the cell. However, for research, diagnostic and therapeutic purposes, there is a need to deliver molecules of interest to the interior of live cells. This is usually accomplished by two methods: (a) carrier mediated delivery which consists of encapsulating the gene/molecule of interest either in an empty viral capsid or in chemically formulated lipoplex or polyplex nanoparticles, or (b) physical methods of delivery, which include the use of different kinds of forces to create reversible pores on the cell membrane (sonoporation, electroporation) or directly inject molecules to the cell cytosol (Gene Gun, microinjection). Of the aforementioned techniques, electroporation is the most commonly used due to it simplicity and ease of use. Our group recently demonstrated a nanochannel based electroporation (NEP) technique, in which two microchannels (˜40 m diameter) are connected by a nanochannel (˜ 200-400 mum diameter) in the center. A cell is positioned in one microchannel such that it makes contact with the nanochannel and transfection agents are placed in the other microchannel. An external electric field applied across the device locally porates the cell where it touches the nancohannel and drives the transfection agents into the cell. Besides maintaining high cell viability and achieving dose control, an important feature of NEP is the delivery of large molecules such as plasmids and quantum dots directly into the cell cytosol. In contrast, delivery of large plasmids during bulk electroporation, wherein cells and genes/plasmids are mixed in a buffered medium and an external electric field is applied across the mixture which electroporates the cells, is via formation of cell membrane bound aggregates which get endocytosed post pulsation. Various mechanisms of DNA transport across the membrane have

  4. In vitro enzymatic studies on the nature and repair of x-ray-induced damages in DNA. Final report

    International Nuclear Information System (INIS)

    Wallace, S.S.

    1981-03-01

    An enzyme has been purified some 4000 fold from Escherichia coli which recognizes alkali stable base damages in x-irradiated DNA. The enzyme has broad specificity incising: DNA damaged by OsO 4 which produces thymine glycols, DNA treated with heat and acid which produces apurinic sites, and DNA uv-irradiated with high fluences which produces a variety of damages including the above. These activities co-chromatograph through Fraction VII the most purified form; however, the optimum reaction parameters differ among the various substrates suggesting the presence of more than one active site. Similar studies have been done with Saccharomyces cerevisiae. Several apurinic activities have been elucidated in this organism, one of which, Endonuclease E, has been purified over 1000 fold. Endonuclease E has been characterized with respect to various reaction parameters as well as by gel electrophoresis. Both the E. coli and yeast enzymes have been used to quantify DNA damage. Apurinic PM2 DNA and OsO 4 -treated PM2 DNA have also been used in a transfection system to estimate the inactivation efficiencies of AP sites and thymine glycols. AP sites have a relatively high inactivation efficiency and contribute about 15% to the inactivation of x-irradiated PM2 phage while thymine glycols contribute significantly less

  5. 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...

  6. 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

  7. Autographa californica multiple nucleopolyhedrovirus DNA polymerase C terminus is required for nuclear localization and viral DNA replication.

    Science.gov (United States)

    Feng, Guozhong; Krell, Peter J

    2014-09-01

    The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral

  8. Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor β1 gene

    International Nuclear Information System (INIS)

    Guo Xiaodong; Zheng Qixin; Yang Shuhua; Shao Zengwu; Yuan Quan; Pan Zhengqi; Tang Shuo; Liu Kai; Quan Daping

    2006-01-01

    Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the combined techniques of gene transfer and tissue engineering. Transforming growth factor beta 1 (TGF-β 1 ) is a multifunctional molecule that plays a central role in promotion of cartilage repair, and inhibition of inflammatory and alloreactive immune response. Cell mediated gene therapy can allow a sustained expression of TGF-β 1 that may circumvent difficulties associated with growth factor delivery. The objective of this study was to investigate whether TGF-β 1 gene modified mesenchymal stem cells (MSCs) could enhance the repair of full-thickness articular cartilage defects in allogeneic rabbits. The pcDNA 3 -TGF-β 1 gene transfected MSCs were seeded onto biodegradable poly-L-lysine coated polylactide (PLA) biomimetic scaffolds in vitro and allografted into full-thickness articular cartilage defects in 18 New Zealand rabbits. The pcDNA 3 gene transfected MSCs/biomimetic scaffold composites and the cell-free scaffolds were taken as control groups I and II, respectively. The follow-up times were 2, 4, 12 and 24 weeks. Macroscopical, histological and ultrastructural studies were performed. In vitro SEM studies found that abundant cartilaginous matrices were generated and completely covered the interconnected pores of the scaffolds two weeks post-seeding in the experimental groups. In vivo, the quality of regenerated tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 24 weeks post-implantation. Joint repair in the experimental groups was better than that of either control group I or II, with respect to: (1) synthesis of hyaline cartilage specific extracellular matrix at the upper portion of the defect; (2) reconstitution of the subchondral bone at the lower portion of the defect and (3) inhibition of inflammatory and alloreactive immune responses. The

  9. Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous {beta}-TCP ceramic scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Guo Xiaodong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Zheng Qixin [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Kulbatski, Iris [Division of Cellular and Molecular Biology, Toronto Western Research Institute, University of Toronto, Toronto, Ontario M5T 2S8 (Canada); Yuan Quan [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Yang Shuhua [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Shao Zengwu [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Wang Hong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Xiao Baojun [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Pan Zhengqi [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China); Tang Shuo [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022 (China)

    2006-09-15

    Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous {beta} tricalcium phosphate ({beta}-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new

  10. Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4

    NARCIS (Netherlands)

    van der Avoort, H. G.; van der Ende, A.; van Arkel, G. A.; Weisbeek, P. J.

    1984-01-01

    The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or

  11. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  12. Epigenetic-based combinatorial resveratrol and pterostilbene alters DNA damage response by affecting SIRT1 and DNMT enzyme expression, including SIRT1-dependent γ-H2AX and telomerase regulation in triple-negative breast cancer

    International Nuclear Information System (INIS)

    Kala, Rishabh; Shah, Harsh N.; Martin, Samantha L.; Tollefsbol, Trygve O.

    2015-01-01

    Nutrition is believed to be a primary contributor in regulating gene expression by affecting epigenetic pathways such as DNA methylation and histone modification. Resveratrol and pterostilbene are phytoalexins produced by plants as part of their defense system. These two bioactive compounds when used alone have been shown to alter genetic and epigenetic profiles of tumor cells, but the concentrations employed in various studies often far exceed physiologically achievable doses. Triple-negative breast cancer (TNBC) is an often fatal condition that may be prevented or treated through novel dietary-based approaches. HCC1806 and MDA-MB-157 breast cancer cells were used as TNBC cell lines in this study. MCF10A cells were used as control breast epithelial cells to determine the safety of this dietary regimen. CompuSyn software was used to determine the combination index (CI) for drug combinations. Combinatorial resveratrol and pterostilbene administered at close to physiologically relevant doses resulted in synergistic (CI <1) growth inhibition of TNBCs. SIRT1, a type III histone deacetylase (HDAC), was down-regulated in response to this combinatorial treatment. We further explored the effects of this novel combinatorial approach on DNA damage response by monitoring γ-H2AX and telomerase expression. With combination of these two compounds there was a significant decrease in these two proteins which might further resulted in significant growth inhibition, apoptosis and cell cycle arrest in HCC1806 and MDA-MB-157 breast cancer cells, while there was no significant effect on cellular viability, colony forming potential, morphology or apoptosis in control MCF10A breast epithelial cells. SIRT1 knockdown reproduced the effects of combinatorial resveratrol and pterostilbene-induced SIRT1 down-regulation through inhibition of both telomerase activity and γ-H2AX expression in HCC1806 breast cancer cells. As a part of the repair mechanisms and role of SIRT1 in recruiting DNMTs

  13. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  14. Regulation of DNA repair by parkin

    International Nuclear Information System (INIS)

    Kao, Shyan-Yuan

    2009-01-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  15. 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...

  16. 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

  17. 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.

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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)

  4. 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

  5. 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.

  6. 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.

  7. Transmission of HCV to a chimpanzee using virus particles produced in an RNA-transfected HepG2 cell culture.

    Science.gov (United States)

    Dash, S; Kalkeri, G; McClure, H M; Garry, R F; Clejan, S; Thung, S N; Murthy, K K

    2001-10-01

    It was demonstrated previously that HepG2 cells produce negative strand RNA and virus-like particles after transfection with RNA transcribed from a full-length hepatitis C virus (HCV) cDNA clone [Dash et al. (1997) American Journal of Pathology, 151:363-373]. To determine in vivo infectivity of these in vitro synthesized viral particles, a chimpanzee was inoculated intravenously with HCV derived from HepG2 cells. The infected chimpanzee was examined serially for elevation of liver enzymes, for the presence of HCV RNA in the serum by reverse transcription nested polymerase chain reaction (RT-PCR), anti-HCV antibodies in the serum, and inflammation in the liver. The chimpanzee developed elevated levels of liver enzymes after the second week, but the levels fluctuated over a 10-week period. HCV RNA was detected in the serum of the chimpanzee at the second, seventh and ninth weeks after inoculation, and remained positive up to 25 weeks. Liver biopsies at Weeks 18 and 19 revealed of mild inflammation. Nucleotide sequence analysis of HCV recovered from the infected chimpanzee at the second and ninth weeks showed 100% sequence homology with the clone used for transfection studies. Serum anti-HCV antibodies were not detected by EIA during the 25 weeks follow-up period. These results suggest that intravenous administration of the virus-like particles derived from RNA-transfected HepG2 cells are infectious, and therefore, the pMO9.6-T7 clone is an infectious clone. These results provide new information that in vitro synthesized HCV particles produced from full-length HCV clone can cause infection in a chimpanzee. This study will facilitate the use of innovative approaches to the study of assembly of HCV particles and mechanisms of virus infectivity in cell culture. Copyright 2001 Wiley-Liss, Inc.

  8. 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)

  9. A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

    Energy Technology Data Exchange (ETDEWEB)

    Oskuee, Reza Kazemi [Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Dosti, Fatemeh [School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Gholami, Leila [Targeted Drug Delivery Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of); Malaekeh-Nikouei, Bizhan, E-mail: malaekehb@mums.ac.ir [Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad (Iran, Islamic Republic of)

    2015-04-01

    Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

  10. A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine

    International Nuclear Information System (INIS)

    Oskuee, Reza Kazemi; Dosti, Fatemeh; Gholami, Leila; Malaekeh-Nikouei, Bizhan

    2015-01-01

    Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65 kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250 nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15 kDa had better transfection efficiency than the vectors made of PAA 65 kDa. In conclusion, results of the present study indicated that grafting PAA 15 kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity. - Highlights: • The modified polyallylamine was synthesized as a gene carrier. • Modification of polyallylamine (15 kDa) with high percentages of hexyl acrylate improved transfection activity remarkably. • Grafting of polymer with acrylate derivatives reduced polymer cytotoxicity especially at percentages of

  11. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J

    1992-01-01

    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...

  12. Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR from transfected cells and placenta explants

    Directory of Open Access Journals (Sweden)

    Randeva Harpal

    2011-05-01

    Full Text Available Abstract Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR. We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm. Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

  13. 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.

  14. 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 m