Enhanced functional recombinant factor VII production by HEK 293 cells stably transfected with VKORC1 where the gamma-carboxylase inhibitor calumenin is stably suppressed by shRNA transfection.

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Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo gamma-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational gamma-carboxylation, the recovery of fully gamma-carboxylated and functional proteins is low. In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K gamma-carboxylase cofactor and in addition stably silenced the gamma-carboxylase inhibitory protein calumenin. Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C.

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

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Lee, Min Sang; Kim, Nak Won; Lee, Kyuri; Kim, Hongtae; Jeong, Ji Hoon

2013-06-01

To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H₂DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

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

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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.

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

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Nakayama, Asuka; Sato, Masahiro; Shinohara, Mariko; Matsubara, Shyuichiro; Yokomine, Takaaki; Akasaka, Eri; Yoshida, Mitsutoshi; Takao, Sonshin

2007-01-01

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.

Transfection of Platyhelminthes

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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.

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

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Lokossou, Adjimon Gatien; Toufaily, Chirine; Vargas, Amandine; Barbeau, Benoit

2016-09-20

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

Transfection in perfused microfluidic cell culture devices: A case study.

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Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

2017-08-01

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

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Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

Proteome alteration induced by hTERT transfection of human fibroblast cells

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Riou Jean-François

2008-04-01

Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions.

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Link, Nils; Brunner, Tobias J; Dreesen, Imke A J; Stark, Wendelin J; Fussenegger, Martin

2007-12-01

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

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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.

Improved biolistic transfection of hair cells.

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Hongyu Zhao

Full Text Available Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C and PMCA2 (ATP2B2; plasma-membrane Ca(2+-ATPase isoform 2 to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

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Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

2006-11-01

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

Evaluation of the magnetic field requirements for nanomagnetic gene transfection

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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

Evaluation of the magnetic field requirements for nanomagnetic gene transfection

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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™.

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

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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.

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

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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�

Transfection in Primary Cultured Neuronal Cells.

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Marwick, Katie F M; Hardingham, Giles E

2017-01-01

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

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

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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. PMID:26274324

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

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Kamel Chettab

Full Text Available 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.

Lipoplex morphologies and their influences on transfection efficiency in gene delivery.

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Ma, Baichao; Zhang, Shubiao; Jiang, Huiming; Zhao, Budiao; Lv, Hongtao

2007-11-20

Cationic lipid-mediated gene transfer is widely used for their advantages over viral gene transfer because it is non-immunogenic, easy to produce and not oncogenic. The main drawback of the application of cationic lipids is their low transfection efficiency. Many reports about transfection efficiency of cationic lipids have been published in recent years. In this review, the current status and prospects for transfection efficiency of different morphologies of lipoplexes are discussed. High transfection activity will be acquired for H(C)(II) structure when membrane fusion is dominant, but when serum is present L(C)(alpha) lipoplexes show great superiority for their inhibition dissociation by serum during lipoplexes transporting. Increasing DOPE often gains high activity for the H(C)(II) structure promoted by DOPE. High lipofection will be gained from large lipoplexes when endocytosis is dominant, because large particles facilitate membrane contact and fusion. We suggest morphologies of lipoplex should be characterized at two levels, lipoplex size and self-assemble structures of lipoplexes, and understanding these would be very important for scientists to prepare novel cationic lipids and design novel formulations with high transfection efficiency.

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

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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

Enhancement of DNA-transfection frequency by X-rays

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Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi [Okayama University Medical School (Japan). Institute of Cellular and Molecular Biology

1997-02-01

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)

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)

Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking.

Science.gov (United States)

Joshee, Nirmal; Bastola, Dhundy R; Cheng, Pi-Wan

2002-11-01

We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.

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.

Optimizing conditions for calcium phosphate mediated transient transfection

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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.

Anchoring cationic amphiphiles for nucleotide delivery: significance of DNA release from cationic liposomes for transfection.

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Hirashima, Naohide; Minatani, Kazuhiro; Hattori, Yoshifumi; Ohwada, Tomohiko; Nakanishi, Mamoru

2007-06-01

We have designed and synthesized lithocholic acid-based cationic amphiphile molecules as components of cationic liposomes for gene transfection (lipofection). To study the relationship between the molecular structures of those amphiphilic molecules, particularly the extended hydrophobic appendant (anchor) at the 3-hydroxyl group, and transfection efficiency, we synthesized several lithocholic and isolithocholic acid derivatives, and examined their transfection efficiency. We also compared the physico-chemical properties of cationic liposomes prepared from these derivatives. We found that isolithocholic acid derivatives exhibit higher transfection efficiency than the corresponding lithocholic acid derivatives. This result indicates that the orientation and extension of hydrophobic regions influence the gene transfection process. Isolithocholic acid derivatives showed a high ability to encapsulate DNA in a compact liposome-DNA complex and to protect it from enzymatic degradation. Isolithocholic acid derivatives also facilitated the release of DNA from the liposome-DNA complex, which is a crucial step for DNA entry into the nucleus. Our results show that the transfection efficiency is directly influenced by the ability of the liposome complex to release DNA, rather than by the DNA-encapsulating ability. Molecular modeling revealed that isolithocholic acid derivatives take relatively extended conformations, while the lithocholic acid derivatives take folded structures. Thus, the efficiency of release of DNA from cationic liposomes in the cytoplasm, which contributes to high transfection efficiency, appears to be dependent upon the molecular shape of the cationic amphiphiles.

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

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...... of the injection site, with a sharply demarcated border between the tumor and brain tissue. In contrast, the parental cell line showed single-cell infiltration and more pronounced destruction of normal brain tissue. Using a 51Cr-release assay, spleen cells from rats transplanted with BT4Cn tumor cells generally...

Transfection of bone marrow derived cells with immunoregulatory proteins.

Science.gov (United States)

Khantakova, Julia N; Silkov, Alexander N; Tereshchenko, Valeriy P; Gavrilova, Elena V; Maksyutov, Rinat A; Sennikov, Sergey V

2018-03-23

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

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Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

1999-01-20

JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA. Copyright 1999

Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency.

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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.

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...

Effect of albumin and dextrose concentration on ultrasound and microbubble mediated gene transfection in vivo.

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Browning, Richard J; Mulvana, Helen; Tang, Meng-Xing; Hajnal, Jo V; Wells, Dominic J; Eckersley, Robert J

2012-06-01

Ultrasound and microbubble mediated gene transfection has great potential for site-selective, safe gene delivery. Albumin-based microbubbles have shown the greatest transfection efficiency but have not been optimised specifically for this purpose. Additionally, few studies have highlighted desirable properties for transfection specific microbubbles. In this article, microbubbles were made with 2% or 5% (w/v) albumin and 20% or 40% (w/v) dextrose solutions, yielding four distinct bubble types. These were acoustically characterised and their efficiency in transfecting a luciferase plasmid (pGL4.13) into female, CD1 mice myocardia was measured. For either albumin concentration, increasing the dextrose concentration increased scattering, attenuation and resistance to ultrasound, resulting in significantly increased transfection. A significant interaction was noted between albumin and dextrose; 2% albumin bubbles made with 20% dextrose showed the least transfection but the most transfection with 40% dextrose. This trend was seen for both nonlinear scattering and attenuation behaviour but not for resistance to ultrasound or total scatter. We have determined that the attenuation behaviour is an important microbubble characteristic for effective gene transfection using ultrasound. Microbubble behaviour can also be simply controlled by altering the initial ingredients used during manufacture. Copyright © 2012 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

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

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......-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.......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...

Cell transfection as a tool to study growth hormone action

DEFF Research Database (Denmark)

Norstedt, G; Enberg, B; Francis, S

1994-01-01

The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration...... 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...

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.

Recombinant protein production from stable mammalian cell lines and pools.

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Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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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

Inhibitory effects of recombinant plasmid pshuttle-Egr1-shTRAIL transfection in combination with X-irradiation on growth of liver cancer cells SMMC7721

International Nuclear Information System (INIS)

Chen Zhiyong; Liu Min; Dong Lihua; Gong Shouliang

2011-01-01

Objective: To investigate the effect of recombinant plasmid pshuttle-Egr1-shTRAIL stable transfection in combination with X-ray irradiation on the TRAIL protein expression and the apoptosis in human SMMC7721 hepatoma cells. Methods: The pshuttle-Egr1-shTRAIL packaged with liposome was stably transfected into SMMC7721 cells in vitro. The shTRAIL protein expression were measured with ELISA assay, Annexin V-FITC kit was adopted to measure the apoptosis of pshuttle-Egr1-shTRAIL cells, and the changes in survival rate of SMMC7721 cells measured with cell cloning assay. Results: The TRAIL protein expressions in pshuttle-Egr1-shTRAIL plus different doses of irradiation groups were significantly increased compared with 0 Gy group (P<0.001). The percentage of apoptotic cells was significantly higher than that in 0 Gy group (P<0.05 or P<0.001), and the survival rate of SMMC7721 cells was decreased significantly (P<0.05 or P<0.001). Conclusion: The pshuttle-Egr1-shTRAIL stable transfection in combination with irradiation can significantly induce the apoptosis of SMMC7721 tumor cells and inhibit the cell proliferation. (authors)

Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA

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Jing Hu

2016-05-01

Full Text Available To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD and a bifunctional R11 (RGD-NLS, which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100–280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18. More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

Optical transfection using an endoscope-like system.

Science.gov (United States)

Ma, Nan; Gunn-Moore, Frank; Dholakia, Kishan

2011-02-01

Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ∼ 6000 cores (pixels) embedded in a fiber cladding of ∼ 300 μm in diameter, produces an image circle (area) of ∼ 270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ∼ 72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

Science.gov (United States)

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression

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

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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

Infectious alphavirus production from a simple plasmid transfection+

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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.

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

Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.

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Masahito Asada

Full Text Available Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd or human dihydrofolate reductase (hdhfr. In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP and human dihydrofolate reductase (hDHFR, was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP and blasticidin-S deaminase (BSD. Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1 gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.

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.

A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

Energy Technology Data Exchange (ETDEWEB)

Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Wan, Min [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Li, Yong-Qiang [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhang, Rong-Ying [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhao, Yuan-Di, E-mail: zydi@mail.hust.edu.cn [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China)

2012-11-15

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.

Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

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Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

2013-09-01

Gene transfection which is the process of deliberately introducing nucleic acids into cells is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure dielectric-barrier discharge (AP-DBD) plasmas. AP-DBD He plasmas are irradiated to the living cell covered with genes. Preliminarily, we use fluorescent dye YOYO-1 instead of the genes and use LIVE/DEAD Stain for cell viability test, and we analyze the transfection efficiency and cell viability under the various conditions. It is clarified that the transfection efficiency is strongly dependence on the plasma irradiation time and cell viability rates is high rates (>90%) regardless of long plasma irradiation time. These results suggest that ROS (Reactive Oxygen Species) and electric field generated by the plasma affect the gene transfection. In addition to this (the plasma irradiation time) dependency, we now investigate the effect of the plasma irradiation under the various conditions.

Multi-lipofection efficiently transfected genes into astrocytes in primary culture.

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Wu, B Y; Liu, R Y; So, K L; Yu, A C

2000-10-30

This study demonstrated that liposome-mediated transfection - lipofection - is suitable for delivering genes into astrocytes. By repeatedly lipofecting the same astrocyte cultures, a process we call multi-lipofection, the transfection efficiency of the beta-galactosidase (beta-gal) gene was improved from 2.6+/-0.6 to 17. 4+/-1.1%. This is the highest efficiency ever reported in gene-transfer with Lipofectin(R) in a primary culture of mouse cerebral cortical astrocytes. Furthermore, multi-lipofection did not cause observable disturbance to astrocytes as indicated by insignificant changes in the glial fibrillary acidic protein content in the cultures. In order to demonstrate that the transfected gene achieved a physiologically relevant expression level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein could be identified intracellularly by immunocytochemistry. Western blot analysis revealed, as compared to astrocytes with one lipofection, a 2.9-fold increase of GDNF with four lipofections. GDNF remained detectable in astrocytes 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a primary culture, making astrocytes good candidate vehicle cells for gene/cell therapy in the CNS.

Unexpected transcellular protein crossover occurs during canonical DNA transfection.

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Arsenault, Jason; Cuijpers, Sabine A G; Niranjan, Dhevahi; Davletov, Bazbek

2014-12-01

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection. © 2014 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

The effect of 17-AAG on iodine uptake kinetics of NIS-transfected anaplastic thyroid cancer

International Nuclear Information System (INIS)

Wang Renfei; Tan Jian; Li Wei; Meng Zhaowei; Zheng Wei

2012-01-01

Objective: To investigate the effect of 17-allylamino-17-demethoxy geldanamycin (17-AAG) on iodine uptake kinetics of NIS-transfected anaplastic thyroid cancer (ATC) cells. Methods: Lipofection was used to transfect the recombinant plasmid, namely pcDNA3.1-NIS, into FRO cells (ATC cell line). A stable cell line NIS-FRO was obtained by G418 resistance selection. 125 I was added into the medium, and influx and efflux experiments were performed. Different time-radioactivity curves were drawn, and further analysis was performed between the non-transfected cells (the control group) and NIS-FRO cells treated with 1 μmol/L 17-AAG for 24 h. Student's t-test was used to analyze the data. Results: The iodine uptake ability of the NIS-FRO cells was significantly higher than that of the FRO cells (about 10.68 times, t=45.329, P<0.001). However, 125 I out-flowed rapidly when removed from the medium, and the retention rate of 125 I in the NIS-FRO cells was only 10.5% of the initial amount after 30 rin. After treatment with 1 μmol/L 17-AAG for 24 h, the 125 I uptake ability of NIS-FRO cells further increased. During the 20-60 min incubation with 125 I, the iodine uptake ability of 17-AAG treated NIS-FRO cells increased significantly with radioactive counts of 31771.8- 54815.5 per minute,which was much higher than that of the control group (24020.3-41293.8 per minute; t=3.096, 4.275, 3.055, 4.292 and 5.496, respectively, all P<0.05). The iodine uptake ability increased about 24.8%-35.5%. Furthermore, 5-30 min after removing the medium, the retention rates of 125 I in the 17-AAG treated NIS-FRO cells were significantly increased compared with those of the control group (32.7%-85.2% vs 10.5%-56.8%; t=22.801, 13.096, 19.631, 38.205, 43.519, 29.322, respectively, all P<0.01), and 125 I efflux was reduced. After 30 min, 125 I retention rate of the treatment group was 32.7%, which was 3.1 times higher than that of the control group. Conclusion: The iodine uptake ability can be

Inhibition of human colorectal adenocarcinoma cells with AdCMV-p53 gene transfection induced by irradiation

International Nuclear Information System (INIS)

Liu Bing; Min Fengling; Xie Yi; Zhou Qingming; Duan Xin; Chinese Academy of Sciences, Beijing; Zhang Hong; Li Wenjian; Hao Jifang; Zhou Guangming; Gao Qingxiang

2006-01-01

The effect of AdCMV-p53 gene transfection induced by γ-ray irradiation on human colorectal adenocarcinoma cells was investigated. The HT-29 cells were irradiated by 0.5, 1.0, 2.0 Gy 60 Co γ-rays, then were transfected with AdCMV-GFP (a replication of deficient recombinant adenoviral vector containing a CMV promoter and green fluorescent protein) or AdCMV-p53 (a replication of deficient recombinant adenoviral vector containing a CMV promoter and carrying human wild p53 gene). Cytotoxity was measured by clonogenic survival assay; apoptosis and the p53 expression were determined by flow cytometry. The results show that the pre-exposure of 0.5 Gy 60 Co γ-rays significantly enhanced the inhibition of HT-29 cells with AdCMV-53 transfection and promoted cell apoptosis. The inhibition rates for the groups of pre-exposure with 0.5 Gy and transfection with 40 and 80 MOI AdCMV-p53 were 50% and 20% higher than those for the groups of the mere transfection, and 40% more than the mere irradiation group. In the case of higher than 0.5 Gy pre-exposure, no significant difference was found between the pre-exposure with transfection group and the mere irradiation group. So 0.5 Gy pre-irradiation and AdCMV-p53 transfection obviously increases the inhibition of HT-29 cells with AdCMV-p53 transfection. The optimum condition is the lower than 1.0 Gy pre-exposure combined with the lower than 80 MOI AdCMV-p53 transfection. (authors)

Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

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Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

2015-12-15

Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

New Transfection Agents Based on Liposomes Containing Biosurfactant MEL-A.

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Nakanishi, Mamoru; Inoh, Yoshikazu; Furuno, Tadahide

2013-08-16

Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs) into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A) are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A.

Graphene Films Show Stable Cell Attachment and Biocompatibility with Electrogenic Primary Cardiac Cells

OpenAIRE

Kim, Taeyong; Kahng, Yung Ho; Lee, Takhee; Lee, Kwanghee; Kim, Do Han

2013-01-01

Graphene has attracted substantial attention due to its advantageous materialistic applicability. In the present study, we tested the biocompatibility of graphene films synthesized by chemical vapor deposition with electrogenic primary adult cardiac cells (cardiomyocytes) by measuring the cell properties such as cell attachment, survival, contractility and calcium transients. The results show that the graphene films showed stable cell attachment and excellent biocompatibility with the electro...

Stable transfection of Eimeria intestinalis and investigation of its life cycle, reproduction and immunogenicity

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Tuanyuan eShi

2016-05-01

Full Text Available Rabbit coccidiosis, caused by infection of Eimeria spp. is one of the most severe parasitic diseases in rabbits. E. intestinalis is one of the most immunogenic species in rabbit coccidia. Due to the lack of genomic information and unsuccessful in vitro cultivation, genetic manipulation of rabbit coccidia lagged behind other apicomplexan parasites. Using regulatory sequences from E. tenella, we obtained a transgenic line of E. intestinalis expressing yellow fluorescent protein (YFP. YFP was continuously expressed throughout the whole life cycle. Morphological features of E. intestinalis in the different developmental stages were dynamically observed with the transgenic line. Some important features in the endogenous development stages were observed. Trophozoites were found as early as 4 h post inoculation. Two-types of schizonts and merozoites were observed in first three of the four schizogonies. Beside jejunum and ileum, gametogony stage and oocysts were also found in the duodenum and vermiform appendix. In addition, the transgenic strain was highly immunogenic but less pathogenic than the wild type. Considering the high immunogenicity of E. intestinalis and amenability to transfection with foreign genes, transgenic E. intestinalis could be a promising oral eukaryotic vaccine vector.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

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Kumar, Ajay; Yellepeddi, Venkata K; Vangara, Kiran K; Strychar, Kevin B; Palakurthi, Srinath

2011-11-01

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

Science.gov (United States)

Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

2016-03-21

The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations.

Efficient transfection of DNA into primarily cultured rat sertoli cells by electroporation.

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Li, Fuping; Yamaguchi, Kohei; Okada, Keisuke; Matsushita, Kei; Enatsu, Noritoshi; Chiba, Koji; Yue, Huanxun; Fujisawa, Masato

2013-03-01

The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P transfection of Sertoli cells.

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

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells.

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Silva, J P Neves; Oliveira, A C N; Casal, M P P A; Gomes, A C; Coutinho, P J G; Coutinho, O P; Oliveira, M E C D Real

2011-10-01

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the

Relationship between the supramolecular structure and the transfection efficiency for cationic micelle/DNA complexes

International Nuclear Information System (INIS)

Sakuragi, Mina; Kusuki, Shota; Hamada, Emi; Sakurai, Kazuo; Masunaga, Hiroyasu; Sasaki, Sono

2009-01-01

We synthesized a cationic lipid benzyl amine derivative bearing a primary amine as the head group and evaluated its transfection efficiency as a DNA carrier. A lipoplex (complex of DNA and lipid micelle) was prepared by mixing BA and two neutral colipids (DOPE and DLPC). When we compared the transfection efficiency at various compositions, we found that B-lipoplex (BA/DOPE/DLPC=1/2/1) was the most efficient while A-lipoplex (BA/DLPC=1/1) showed no transfection. We compared A-lipoplex with B-lipoplex by use of SAXS, fluorescence spectrum of ethidium bromide and pyrene. These results indicated that A-lipoplex formed a lamellar or cylinder structure within which DNA molecules were trapped in the lipid alkyl chain, while B-lipoplex formed cylinders where DNAs were intercalated between the lipid micelle cylinders. (author)

New Transfection Agents Based on Liposomes Containing Biosurfactant MEL-A

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Tadahide Furuno

2013-08-01

Full Text Available Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A.

[EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

Science.gov (United States)

Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

2015-04-01

To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into

Effects of molecular size and chemical factor on plasma gene transfection

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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.

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

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Lummis Sarah CR

2011-06-01

Full Text Available Abstract Background Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles. Results Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles. Conclusions We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.

mRNA transfection of mouse and human neural stem cell cultures.

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Samuel McLenachan

Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

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McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

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McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability.

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Kraus, Armin; Täger, Joachim; Kohler, Konrad; Haerle, Max; Werdin, Frank; Schaller, Hans-Eberhard; Sinis, Nektarios

2010-11-01

To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC). The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined. Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-L-ornithine coated plates was compromised by either method. Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

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Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

2017-06-02

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

2014-03-01

Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

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.

Promoter, transgene, and cell line effects in the transfection of mammalian cells using PDMAEMA-based nano-stars

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Alexander Raup

2016-09-01

Full Text Available Non-viral transfection protocols are typically optimized using standard cells and reporter proteins, potentially underestimating cellular or transgene effects. Here such effects were studied for two human (Jurkat, HEK-293 and two rodent (CHO-K1, L929 cell lines and three fluorescent reporter proteins. Expression of the enhanced green fluorescent protein (EGFP was studied under the control of the human elongation factor 1 alpha promoter and three viral promoters (SV40, SV40/enhancer, CMV, that of ZsYellow1 (yellow fluorescence and mCherry (red fluorescence for the CMV promoter. Results varied with the cell line, in particular for the Jurkat cells. Pair-wise co-transfection of the CMV controlled transgenes resulted in a significant fraction of monochromatic cells (EGFP for EGFP/YFP and EGFP/RFP co-transfections, YFP in case of YFP/RFP co-transfections. Only Jurkat cells were almost incapable of expressing YFP. Dilution of the plasmid DNA with a non-expressed plasmid showed cell line dependent effects on transfection efficiency and/or expression levels.

Experimental Model of Gene Transfection in Healthy Canine Myocardium: Perspectives of Gene Therapy for Ischemic Heart Disease

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Renato A. K. Kalil

2002-09-01

Full Text Available OBJECTIVE: To assess the transfection of the gene that encodes green fluorescent protein (GFP through direct intramyocardial injection. METHODS: The pREGFP plasmid vector was used. The EGFP gene was inserted downstream from the constitutive promoter of the Rous sarcoma virus. Five male dogs were used (mean weight 13.5 kg, in which 0.5 mL of saline solution (n=1 or 0.5 mL of plasmid solution containing 0.5 µg of pREGFP/dog (n=4 were injected into the myocardium of the left ventricular lateral wall. The dogs were euthanized 1 week later, and cardiac biopsies were obtained. RESULTS: Fluorescence microscopy showed differences between the cells transfected and not transfected with pREGFP plasmid. Mild fluorescence was observed in the cardiac fibers that received saline solution; however, the myocardial cells transfected with pREGFP had overt EGFP expression. CONCLUSION: Transfection with the EGFP gene in healthy canine myocardium was effective. The reproduction of this efficacy using vascular endothelial growth factor (VEGF instead of EGFP aims at developing gene therapy for ischemic heart disease.

Off-resonance plasmonic enhanced femtosecond laser optoporation and transfection of cancer cells.

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Baumgart, Judith; Humbert, Laure; Boulais, Étienne; Lachaine, Rémi; Lebrun, Jean-Jaques; Meunier, Michel

2012-03-01

A femtosecond laser based transfection method using off-resonance plasmonic gold nanoparticles is described. For human cancer melanoma cells, the treatment leads to a very high perforation rate of 70%, transfection efficiency three times higher than for conventional lipofection, and very low toxicity (transfection for skin cancer treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Noninvasive imaging of transplanted living functional cells transfected with a reporter estrogen receptor gene

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Takamatsu, Shinji [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)]. E-mail: shinjit@fmsrsa.fukui-med.ac.jp; Furukawa, Takako [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Yonekura, Yoshiharu [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Yoshida, Fukui 910-1193 (Japan)

2005-11-01

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 16{alpha}-[{sup 18}F]-fluoro-17{beta}-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 [{sup 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 [{sup 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.

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

Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients

International Nuclear Information System (INIS)

Kibitel, J.T.; Yee, V.; Yarosh, D.B.

1991-01-01

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells. (author)

Cationic Phospholipids Forming Cubic Phases: Lipoplex Structure and Transfection Efficiency

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Koynova, Rumiana; Wang, Li; MacDonald, Robert C. (NWU)

2008-10-29

The transfection activity and the phase behavior of two novel cationic O-alkyl-phosphatidylcholines, 1,2-dioleoyl-sn-glycero-3-hexylphosphocholine (C6-DOPC) and 1,2-dierucoyl-sn-glycero-3-ethylphosphocholine (di22:1-EPC), have been examined with the aim of more completely understanding the mechanism of lipid-mediated DNA delivery. Both lipids form cubic phases: C6-DOPC in the entire temperature range from -10 to 90 C, while di22:1-EPC exhibits an irreversible lamellar-cubic transition between 50 and 70 C on heating. The lipoplexes formed by C6-DOPC arrange into hexagonal phase, while the lipoplexes of di22:1-EPC are lamellar. Both lipids exhibit lower transfection activity than the lamellar-forming 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC). Thus, for the studied cationic phospholipid-DNA systems, the lipoplex phase state is a factor that does not seem to correlate with transfection activity. The parameter that exhibits better correlation with the transfection activity within the present data set is the phase state of the lipid dispersion prior to the addition of DNA. Thus, the lamellar lipid dispersion (EDOPC) produces more efficient lipoplexes than the dispersion with coexisting lamellar and cubic aggregates (diC22:1-EPC), which is even more efficient than the purely cubic dispersions (C6-DOPC; diC22:1-EPC after heating). It could be inferred from these data and from previous research that cubic phase lipid aggregates are unlikely to be beneficial to transfection. The lack of correlation between the phase state of lipoplexes and their transfection activity observed within the present data set does not mean that lipid phase state is generally unimportant for lipofection: a viewpoint now emerging from our previous studies is that the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids.

Cationic phospholipids forming cubic phases: lipoplex structure and transfection efficiency.

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Koynova, Rumiana; Wang, Li; Macdonald, Robert C

2008-01-01

The transfection activity and the phase behavior of two novel cationic O-alkyl-phosphatidylcholines, 1,2-dioleoyl- sn-glycero-3-hexylphosphocholine (C6-DOPC) and 1,2-dierucoyl- sn-glycero-3-ethylphosphocholine (di22:1-EPC), have been examined with the aim of more completely understanding the mechanism of lipid-mediated DNA delivery. Both lipids form cubic phases: C6-DOPC in the entire temperature range from -10 to 90 degrees C, while di22:1-EPC exhibits an irreversible lamellar-cubic transition between 50 and 70 degrees C on heating. The lipoplexes formed by C6-DOPC arrange into hexagonal phase, while the lipoplexes of di22:1-EPC are lamellar. Both lipids exhibit lower transfection activity than the lamellar-forming 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine (EDOPC). Thus, for the studied cationic phospholipid-DNA systems, the lipoplex phase state is a factor that does not seem to correlate with transfection activity. The parameter that exhibits better correlation with the transfection activity within the present data set is the phase state of the lipid dispersion prior to the addition of DNA. Thus, the lamellar lipid dispersion (EDOPC) produces more efficient lipoplexes than the dispersion with coexisting lamellar and cubic aggregates (diC22:1-EPC), which is even more efficient than the purely cubic dispersions (C6-DOPC; diC22:1-EPC after heating). It could be inferred from these data and from previous research that cubic phase lipid aggregates are unlikely to be beneficial to transfection. The lack of correlation between the phase state of lipoplexes and their transfection activity observed within the present data set does not mean that lipid phase state is generally unimportant for lipofection: a viewpoint now emerging from our previous studies is that the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

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Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Role of cholesterol on the transfection barriers of cationic lipid/DNA complexes

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Pozzi, Daniela; Cardarelli, Francesco; Salomone, Fabrizio; Marchini, Cristina; Amenitsch, Heinz; Barbera, Giorgia La; Caracciolo, Giulio

2014-08-01

Most lipid formulations need cholesterol for efficient transfection, but the precise motivation remains unclear. Here, we have investigated the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphocholine in Chinese hamster ovary cells. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by TE, synchrotron small angle X-ray scattering, and laser scanning confocal microscopy experiments. We prove that cholesterol-containing lipoplexes enter the cells using different endocytosis pathways. Formulations with high cholesterol content efficiently escape from endosomes and exhibit a lamellar-nonlamellar phase transition in mixture with biomembrane mimicking lipid formulations. This might explain both the DNA release ability and the high transfection efficiency. These studies highlight the enrichment in cholesterol as a decisive factor for transfection and will contribute to the rational design of lipid nanocarriers with superior TE.

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

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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.

Design, synthesis, and in vitro transfection biology of novel tocopherol based monocationic lipids: a structure-activity investigation.

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Kedika, Bhavani; Patri, Srilakshmi V

2011-01-27

Herein, we report on the design, synthesis, and in vitro gene delivery efficacies of five novel tocopherol based cationic lipids (1-5) in transfecting CHO, B16F10, A-549, and HepG2 cells. The in vitro gene transfer efficiencies of lipids (1-5) were evaluated by both β-galactosidase reporter gene expression and inverted fluorescent microscopic experiments. The results of the present structure-activity investigation convincingly demonstrate that the tocopherol based lipid with three hydroxyl groups in its headgroup region showed 4-fold better transfection efficiency than the commercial formulation. The results also demonstrate that these tocopherol based lipids may be targeted to liver. Transfection efficiency of all the relevant lipids was maintained even when the serum was present during the transfection conditions. The results indicated that the designed systems are quite capable of transferring the DNA into all four types of cells studied with low or no toxicity.

Cellular Injury of Cardiomyocytes during Hepatocyte Growth Factor Gene Transfection with Ultrasound-Triggered Bubble Liposome Destruction

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Kazuo Komamura

2011-01-01

Full Text Available We transfected naked HGF plasmid DNA into cultured cardiomyocytes using a sonoporation method consisting of ultrasound-triggered bubble liposome destruction. We examined the effects on transfection efficiency of three concentrations of bubble liposome (1×106, 1×107, 1×108/mL, three concentrations of HGF DNA (60, 120, 180 μg/mL, two insonification times (30, 60 sec, and three incubation times (15, 60, 120 min. We found that low concentrations of bubble liposome and low concentrations of DNA provided the largest amount of the HGF protein expression by the sonoporated cardiomyocytes. Variation of insonification and incubation times did not affect the amount of product. Following insonification, cardiomyocytes showed cellular injury, as determined by a dye exclusion test. The extent of injury was most severe with the highest concentration of bubble liposome. In conclusion, there are some trade-offs between gene transfection efficiency and cellular injury using ultrasound-triggered bubble liposome destruction as a method for gene transfection.

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.

Uptake of DNA by cancer cells without a transfection reagent

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Yanping Kong

Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

OpenAIRE

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize deliver...

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

OpenAIRE

Dong, Yuzhen; Yang, Libin; Yang, Lin; Zhao, Hongxing; Zhang, Chao; Wu, Dapeng

2014-01-01

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesenchymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal ...

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

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Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

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Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

Non-viral transfection methods optimized for gene delivery to a lung cancer cell line.

Science.gov (United States)

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-04-01

Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods.

Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

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Thomas Wurster

Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

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Ana V Oliveira

2013-01-01

Full Text Available Objective: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. Materials and Methods: Chitosan and thiolated chitosan nanoparticles (NPs were prepared in order to obtain a NH3 + :PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. Results: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. Conclusion: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.

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

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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.

Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

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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.

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

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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 ...

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

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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.

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

International Nuclear Information System (INIS)

Steitz, Benedikt; Hofmann, Heinrich; Kamau, Sarah W.; Hassa, Paul O.; Hottiger, Michael O.; Rechenberg, Brigitte von; Hofmann-Amtenbrink, Magarethe; Petri-Fink, Alke

2007-01-01

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, ζ-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

Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

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Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

2014-05-01

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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)

Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

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Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

2014-02-03

Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

DEFF Research Database (Denmark)

Vinther, Jeppe; Hedegaard, Mads Marquardt; Gardner, Paul Phillip

2006-01-01

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection...

Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

International Nuclear Information System (INIS)

Zhao Xiaoli; Li Zhaoyang; Liu Wenguang; Lam, Wingmoon; Sun Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

2011-01-01

Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R 8 -CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R 8 -CSs were characterized using FTIR and 1 H NMR. Agarose gel electrophoresis assay showed that R 8 -CS could efficiently condense the DNA. The particle size of R 8 -CS/DNA complexes were determined to be around 100–200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R 8 -CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

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)

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

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Qin, Lu; Huanzhang, Niu; Guangyu, Zhu; Yanli, An; Dinghong, Qiu; Gaojun, Teng [Radiologic Department, Zhongda Hospital, Southeast Univ., Nanjing (China)

2007-02-15

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 {mu}g)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 {mu}g, 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)

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

Science.gov (United States)

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into

Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

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Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

2016-01-01

This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

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.

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.

Characterization of cell lines stably transfected with rubella virus replicons

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

2012-07-20

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

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

International Nuclear Information System (INIS)

Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

1987-01-01

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

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.

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; Murua Escobar, Hugo; 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.

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

Structure relationship of cationic lipids on gene transfection mediated by cationic liposomes.

Science.gov (United States)

Paecharoenchai, Orapan; Niyomtham, Nattisa; Apirakaramwong, Auayporn; Ngawhirunpat, Tanasait; Rojanarata, Theerasak; Yingyongnarongkul, Boon-ek; Opanasopit, Praneet

2012-12-01

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.

Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

Science.gov (United States)

Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

2004-11-01

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

Green fluorescent protein as indicator of nonviral transient transfection efficiency in endometrial and testicular biopsies.

Science.gov (United States)

Zizzi, Antonio; Minardi, Daniele; Ciavattini, Andrea; Giantomassi, Federica; Montironi, Rodolfo; Muzzonigro, Giovanni; Di Primio, Roberto; Lucarini, Guendalina

2010-03-01

In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.

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

Directory of Open Access Journals (Sweden)

Lin-Lin Liu

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

Nonviral transfection of adipose tissue stromal cells: an experimental study.

Science.gov (United States)

Lopatina, T V; Kalinina, N I; Parfyonova, E V

2009-04-01

Delivery of plasmid DNA and interfering RNA into adipose tissue stromal cells was carried out by the methods of lipofection, calcium phosphate method, and by electroporation. The percent of transfected cells after delivery of plasmid DNA by the calcium phosphate method and lipofection was 0 and 15%, respectively, vs. more than 50% after electroporation. Similar results were obtained for delivery of short-strand RNA into cells. These data indicate that electroporation is the most effective method of nonviral transfection of adipose tissue stromal cells.

Hyperbranched–dendrimer architectural copolymer gene delivery using hyperbranched PEI conjugated to poly(propyleneimine) dendrimers: synthesis, characterization, and evaluation of transfection efficiency

Energy Technology Data Exchange (ETDEWEB)

Alavi, Seyyed Jamal [Ferdowsi University of Mashhad, Department of Chemistry, Faculty of Science (Iran, Islamic Republic of); Gholami, Leila [Mashhad University of Medical Sciences, Department of Modern Sciences and Technologies, School of Medicine (Iran, Islamic Republic of); Askarian, Saeedeh [Mashhad University of Medical Sciences, Department of Medical Biotechnology, School of Medicine (Iran, Islamic Republic of); Darroudi, Majid [Mashhad University of Medical Sciences, Nuclear Medicine Research Center (Iran, Islamic Republic of); Massoudi, Abdolhossein [University of Payam noor, Department of Chemistry (Iran, Islamic Republic of); Rezaee, Mehdi; Kazemi Oskuee, Reza, E-mail: Oskueekr@mums.ac.ir [Mashhad University of Medical Sciences, Department of Medical Biotechnology, School of Medicine (Iran, Islamic Republic of)

2017-02-15

The applications of dendrimer-based vectors seem to be promising in non-viral gene delivery because of their potential for addressing the problems with viral vectors. In this study, generation 3 poly(propyleneimine) (G3-PPI) dendrimers with 1, 4-diaminobutane as a core initiator was synthesized using a divergent growth approach. To increase the hydrophobicity and reduce toxicity, 10% of primary amines of G3-PPI dendrimers were replaced with bromoalkylcarboxylates with different chain lengths (6-bromohexanoic and 10-bromodecanoic). Then, to retain the overall buffering capacity and enhance transfection, the alkylcarboxylate–PPIs were conjugated to 10 kDa branched polyethylenimine (PEI). The results showed that the modified PPI was able to form complexes with the diameter of less than 60 nm with net-positive surface charge around 20 mV. No significant toxicity was observed in modified PPIs; however, the hexanoate conjugated PPI–PEI (PPI-HEX-10% PEI) and the decanoate conjugated PPI–PEI (PPI-DEC-10%-PEI) showed the best transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, even PPI-HEX-10%-PEI showed transfection efficiency equal to standard PEI 25 kDa with reduced toxicity. This study suggested a new series of hyperbranched (PEI)–dendrimer (PPI) architectural copolymers as non-viral gene delivery vectors with high transfection efficiency and low toxicity.

Hyperbranched-dendrimer architectural copolymer gene delivery using hyperbranched PEI conjugated to poly(propyleneimine) dendrimers: synthesis, characterization, and evaluation of transfection efficiency

Science.gov (United States)

Alavi, Seyyed Jamal; Gholami, Leila; Askarian, Saeedeh; Darroudi, Majid; Massoudi, Abdolhossein; Rezaee, Mehdi; Kazemi Oskuee, Reza

2017-02-01

The applications of dendrimer-based vectors seem to be promising in non-viral gene delivery because of their potential for addressing the problems with viral vectors. In this study, generation 3 poly(propyleneimine) (G3-PPI) dendrimers with 1, 4-diaminobutane as a core initiator was synthesized using a divergent growth approach. To increase the hydrophobicity and reduce toxicity, 10% of primary amines of G3-PPI dendrimers were replaced with bromoalkylcarboxylates with different chain lengths (6-bromohexanoic and 10-bromodecanoic). Then, to retain the overall buffering capacity and enhance transfection, the alkylcarboxylate-PPIs were conjugated to 10 kDa branched polyethylenimine (PEI). The results showed that the modified PPI was able to form complexes with the diameter of less than 60 nm with net-positive surface charge around 20 mV. No significant toxicity was observed in modified PPIs; however, the hexanoate conjugated PPI-PEI (PPI-HEX-10% PEI) and the decanoate conjugated PPI-PEI (PPI-DEC-10%-PEI) showed the best transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, even PPI-HEX-10%-PEI showed transfection efficiency equal to standard PEI 25 kDa with reduced toxicity. This study suggested a new series of hyperbranched (PEI)-dendrimer (PPI) architectural copolymers as non-viral gene delivery vectors with high transfection efficiency and low toxicity.

Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

NARCIS (Netherlands)

van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

Energy Technology Data Exchange (ETDEWEB)

Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Nishimura, Kohji [Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Jisaka, Mitsuo; Nagaya, Tsutomu [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Shono, Fumiaki [Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514 (Japan); Yokota, Kazushige, E-mail: yokotaka@life.shimane-u.ac.jp [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan)

2012-02-15

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

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.

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

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)

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

DEFF Research Database (Denmark)

Wu, Kaimin; Xu, Jie; Liu, Mingzhe

2013-01-01

MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes...... of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection...... formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing...

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.

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

NARCIS (Netherlands)

Kneuer, C; Sameti, M; Bakowsky, U; Schiestel, T; Schirra, H

2000-01-01

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection

Transfection Agent Induced Nanoparticle Cell Loading

Directory of Open Access Journals (Sweden)

Karin Montet-Abou

2005-07-01

Full Text Available Loading cells with magnetic nanoparticles, and tracking their fate in vivo by high resolution MRI, is an attractive approach for enhancing the efficacy of cell-based therapies including those utilizing hematopoietic stem cells, neuroprogenitor cells, and T cells. The transfection agent (internalization agent assisted loading with the Feridex IV® nanoparticle is an attractive method of loading because of the low cost of materials, and possible low regulatory barriers for eventual clinical use. We therefore explored the interaction between Feridex IV® and three internalization agents protamine (PRO, polylysine (PLL, and lipofectamine (LFA. Feridex reacted with internalization agents to form aggregates, except when either the internalization agent or Feridex was present in large excess. When Jurkat T cells were incubated with Feridex/LFA or Feridex/PRO mixtures, and washed by centrifugation, nanoparticle aggregates co-purified with cells. With C17.2 cells large iron oxide particles adhered to the cell surface. At 30 μg/mL Feridex and 3 μg/mL LFA, internalization was largely mediated by LFA and was largely cytoplasmic. However, we found that the conditions used to label cells with Feridex and transfection agents need to be carefully selected to avoid the problems of surface adsorption and nanoparticle precipitation.

Optical sorting and photo-transfection of mammalian cells

CSIR Research Space (South Africa)

Mthunzi, P

2010-02-01

Full Text Available and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human...

Establishing malaria parasite transfection technology in South Africa

CSIR Research Space (South Africa)

Van Brummelen, AC

2010-01-01

Full Text Available -richness and intracellular location of the organism. As a result such successful transfection often requires prolonged periods (up to 2-3 months) of constant and patient culturing and selection. In addition, plasmids usually have a complicated composition and require lengthy...

Restoration of u.v.-induced excision repair in Xeroderma D cells transfected with the denV gene of bacteriophage T4

International Nuclear Information System (INIS)

Arrand, J.E.; Squires, S.; Bone, N.M.; Johnson, R.T.

1987-01-01

The heritable DNA repair defect in human Xeroderma D cells, resulting in failure to incise at u.v. light-induced pyrimidine dimers, has been partially but stably corrected by transfection of immortalised cells with the denV pyrimidine dimer glycosylase gene of bacteriophage T4. Transfectants selected either for a dominant marker on the mammalian vector carrying the prokaryotic gene or for dominant marker plus resistance to killing by u.v. light, were shown to express the denV gene to varying degrees. denV expression results in significant phenotypic change in the initially repair-deficient, u.v.-hypersensitive cells. Increased resistance to u.v. light and more rapid recovery of replicative DNA synthesis following u.v. irradiation were correlated with improved repair DNA synthesis and with a novel dimer incision capability present in denV transfected Xeroderma cells but not as evident in transfected normal cells. Most transfectants contain a single integrated copy of the denV gene; increase in denV copy number does not result in either increased gene expression or enhanced survival to u.v. light. Results show that expression of a heterologous prokaryotic repair gene can partially compensate for the genetic defect in a human Xeroderma D cell. (author)

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

Science.gov (United States)

Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

2016-04-01

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

Towards gene therapy based on femtosecond optical transfection

Science.gov (United States)

Antkowiak, M.; Torres-Mapa, M. L.; McGinty, J.; Chahine, M.; Bugeon, L.; Rose, A.; Finn, A.; Moleirinho, S.; Okuse, K.; Dallman, M.; French, P.; Harding, S. E.; Reynolds, P.; Gunn-Moore, F.; Dholakia, K.

2012-06-01

Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

Lethal effects of 32P decay on transfecting activity of Bacillus subtillis phage phie DNA

International Nuclear Information System (INIS)

Loveday, K.S.

1979-01-01

Disintegration of 32 P present in the DNA of Bacillus subtilis phage phie (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 1/12 of the 32 P disintegrations per phage DNA equivalent inactivities the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by phie DNA

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.

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

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Min, Jung-Jun; Iyer, Meera [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Gambhir, Sanjiv S. [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Department of Radiology, Bio-X Program, Stanford University, Stanford, California (United States)

2003-11-01

Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells - stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [{sup 18}F]FHBG, [{sup 3}H]PCV, and [{sup 14}C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [{sup 14}C]FIAU accumulation was greater than that of [{sup 3}H]PCV and [{sup 18}F]FHBG in control cells and in HSV1-tk stably transfected cells (P<0.001). After infection of C6 cells with AdCMV-HSV1-tk (1.5 x 10{sup 8} pfu), [{sup 18}F]FHBG and [{sup 3}H]PCV accumulation was significantly greater than that of [{sup 14}C]FIAU (P<0.01). [{sup 18}F]FHBG and [{sup 3}H]PCV accumulated to a significantly greater extent than [{sup 14}C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P<0.001). Results from the nude mice supported the results in cell culture. [{sup 14}C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [{sup 18}F]FHBG (P<0.05); however, compared with [{sup 14}C]FIAU, [{sup 18}F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [{sup 18}F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A

Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

Science.gov (United States)

Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

2016-01-01

. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals. PMID:27911903

Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.

Directory of Open Access Journals (Sweden)

Daiane P Oldiges

2016-12-01

GST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln B. bovis parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of R. microplus feeding in experimentally inoculated animals.

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.

Synthesis and in vitro transfection efficiency of spermine-based cationic lipids with different central core structures and lipophilic tails.

Science.gov (United States)

Niyomtham, Nattisa; Apiratikul, Nuttapon; Suksen, Kanoknetr; Opanasopit, Praneet; Yingyongnarongkul, Boon-Ek

2015-02-01

Twelve spermine-based cationic lipids with four different central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) and three hydrophobic tails (lauric acid, myristic acid and palmitic acid) were synthesized. The liposomes containing lipids and DOPE showed moderate to good in vitro DNA delivery into HeLa cells. GFP expression experiments revealed that liposomes composed of lipids with 3-amino-1,2-dioxypropyl as a central core structure exhibited highest transfection efficiency under serum-free condition. Whereas, lipid with 2-amino-1,3-dioxypropyl core structure showed highest transfection under 10% serum condition. Moreover, the liposomes and lipoplexes composted of these cationic lipids exhibited low cytotoxicity. Copyright © 2015. Published by Elsevier Ltd.

Mannosylated Chitosan Nanoparticles Based Macrophage-Targeting Gene Delivery System Enhanced Cellular Uptake and Improved Transfection Efficiency.

Science.gov (United States)

Peng, Yixing; Yao, Wenjun; Wang, Bo; Zong, Li

2015-04-01

Gene transfer mediated by mannosylated chitosan (MCS) is a safe and promising approach for gene and vaccine delivery. MCS nanoparticles based gene delivery system showed high in vivo delivery efficiency and elicited strong immune responses in mice. However, little knowledge about the cell binding, transfection efficiency and intracellular trafficking of MCS nanoparticles had been acquired. In this study, using gastrin-releasing peptide as a model plasmid (pGRP), the binding of MCS/pGRP nanoparticles to macrophages and the intracellular trafficking of MCS/pGRP nanoparticles in macrophages were investigated. MCS-mediated transfection efficiency in macrophages was also evaluated using pGL-3 as a reporter gene. The results showed that the binding and transfection efficiency of MCS nanoparticles in macrophages was higher than that of CS, which was attributed to the interaction between mannose ligands in MCS and mannose receptors on the surface of macrophages. Observation with a confocal laser scanning microscope indicated the cellular uptake of MCS/pGRP nanoparticles were more than that of CS/pGRP nanoparticles in macrophages. MCS/pGRP nanoparticles were taken up by macrophages and most of them were entrapped in endosomal/lysosomal compartments. After the nanoparticles escaping from endosomal/lysosomal compartments, naked pGRP entered the nucleus, and a few MCS might enter the nucleus in terms of nanoparticles. Overall, MCS has the potential to be an excellent macrophage-targeting gene delivery carrier.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury.

Science.gov (United States)

Dong, Yuzhen; Yang, Libin; Yang, Lin; Zhao, Hongxing; Zhang, Chao; Wu, Dapeng

2014-08-15

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesenchymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

Science.gov (United States)

2011-01-01

Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells. PMID:21663599

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

Directory of Open Access Journals (Sweden)

Lisowska Katarzyna Marta

2011-06-01

Full Text Available Abstract Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1 gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA, Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i some cationic liposomes may not be suitable for functional studies on hsp promoters, ii lipofection may cause unintended changes in global gene expression in the transfected cells.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

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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

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

Science.gov (United States)

Campos, Vinicius Farias; de Leon, Priscila Marques Moura; Komninou, Eliza Rossi; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

2011-11-01

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT. Copyright © 2011 Elsevier Inc. All rights reserved.

Mechanism of attenuation of a chimeric influenza A/B transfectant virus.

Science.gov (United States)

Luo, G; Bergmann, M; Garcia-Sastre, A; Palese, P

1992-08-01

The ribonucleoprotein transfection system for influenza virus allowed us to construct an influenza A virus containing a chimeric neuraminidase (NA) gene in which the noncoding sequence is derived from the NS gene of influenza B virus (T. Muster, E. K. Subbarao, M. Enami, B. P. Murphy, and P. Palese, Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991). This transfectant virus is attenuated in mice and grows to lower titers in tissue culture than wild-type virus. Since such a virus has characteristics desirable for a live attenuated vaccine strain, attempts were made to characterize this virus at the molecular level. Our analysis suggests that the attenuation of the virus is due to changes in the cis signal sequences, which resulted in a reduction of transcription and replication of the chimeric NA gene. The major finding concerns a sixfold reduction in NA-specific viral RNA in the virion, causing a reduction in the ratio of infectious particles to physical particles compared with the ratio in wild-type virus. Although the NA-specific mRNA level is also reduced in transfectant virus-infected cells, it does not appear to contribute to the attenuation characteristics of the virus. The levels of the other RNAs and their expression appear to be unchanged for the transfectant virus. It is suggested that downregulation of the synthesis of one viral RNA segment leads to the generation of defective viruses during each replication cycle. We believe that this represents a general principle for attenuation which may be applied to other segmented viruses containing either single-stranded or double-stranded RNA.

Protocol for Lipid-Mediated Transient Transfection in A549 Epithelial Lung Cell Line.

Science.gov (United States)

Marcos-Vadillo, Elena; García-Sánchez, Asunción

2016-01-01

Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. They have been used in a wide range of research fields. In this chapter, a method of transient transfection of the A549 cell line, human lung cells of alveolar epithelium, with an expression plasmid is described. In addition, the fundamental characteristics of this experimental procedure are addressed.

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

Energy Technology Data Exchange (ETDEWEB)

Amand, Helene L., E-mail: helene.amand@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Norden, Bengt, E-mail: norden@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Fant, Kristina, E-mail: kristina.fant@sp.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden)

2012-02-17

Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer 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

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.

Pharmaceutical studies for gene therapy: expression of human Cu, Zn-superoxide dismutase gene transfected by lipofection in rat skin fibroblasts.

Science.gov (United States)

Nishiguchi, K; Ishida, K; Nakajima, M; Maeda, T; Komada, F; Iwakawa, S; Tanigawara, Y; Okumura, K

1996-08-01

To evaluate whether lipofection using Lipofectin is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts. However, cytotoxic effects in both skin fibroblasts were observed with high doses of Lipofectin. On the other hand, with optimal amounts of DNA and Lipofectin, the reporter gene (NeoT) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin for each cell type, and that transfection with Lipofectin under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.

Cationic nanoparticles with quaternary ammonium-functionalized PLGA–PEG-based copolymers for potent gene transfection

Energy Technology Data Exchange (ETDEWEB)

Wang, Yan-Hsung [Kaohsiung Medical University, School of Dentistry, College of Dental Medicine (China); Fu, Yin-Chih [Kaohsiung Medical University, Graduate Institute of Medicine, College of Medicine (China); Chiu, Hui-Chi [Kaohsiung Medical University, Department of Medicinal and Applied Chemistry, College of Life Science (China); Wang, Chau-Zen [Kaohsiung Medical University, Department of Physiology, College of Medicine (China); Lo, Shao-Ping [Kaohsiung Medical University, Department of Medicinal and Applied Chemistry, College of Life Science (China); Ho, Mei-Ling [Kaohsiung Medical University, Department of Physiology, College of Medicine (China); Liu, Po-Len [Kaohsiung Medical University, Department of Respiratory Therapy, College of Medicine (China); Wang, Chih-Kuang, E-mail: ckwang@kmu.edu.tw [Kaohsiung Medical University, Department of Medicinal and Applied Chemistry, College of Life Science (China)

2013-11-15

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{sub 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.

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.

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

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

Science.gov (United States)

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Epizone: Interlaboratory Ring Trial to Compare Dna Transfection Efficiencies

DEFF Research Database (Denmark)

Dory, Daniel; Albina, Emmanuel; Kwiatek, Olivier

Chemical-based transfection of DNA into cultured cells is routinely used to study for example viral or cellular gene functions involved in virus replication, to analyse cellular defence mechanisms or develop specific strategies to interfere with virus replication. Other applications include rescu...

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

Science.gov (United States)

Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. PMID:25364253

Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.

Science.gov (United States)

Villa-Diaz, Luis G; Garcia-Perez, Jose L; Krebsbach, Paul H

2010-12-01

Because human embryonic stem (hES) cells can differentiate into virtually any cell type in the human body, these cells hold promise for regenerative medicine. The genetic manipulation of hES cells will enhance our understanding of genes involved in early development and will accelerate their potential use and application for regenerative medicine. The objective of this study was to increase the transfection efficiency of plasmid DNA into hES cells by modifying a standard reverse transfection (RT) protocol of lipofection. We hypothesized that immobilization of plasmid DNA in extracellular matrix would be a more efficient method for plasmid transfer due to the affinity of hES cells for substrates such as Matrigel and to the prolonged exposure of cells to plasmid DNA. Our results demonstrate that this modification doubled the transfection efficiency of hES cells and the generation of clonal cell lines containing a piece of foreign DNA stably inserted in their genomes compared to results obtained with standard forward transfection. In addition, treatment with dimethyl sulfoxide further increased the transfection efficiency of hES cells. In conclusion, modifications to the RT protocol of lipofection result in a significant and robust increase in the transfection efficiency of hES cells.

BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

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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.

Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine

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Jennifer A. Fortune

2011-01-01

Full Text Available We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp. while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.

The density of GM1-enriched lipid rafts correlates inversely with the efficiency of transfection mediated by cationic liposomes.

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Kovács, Tamás; Kárász, Andrea; Szöllosi, János; Nagy, Peter

2009-08-01

Although cationic liposome-mediated transfection has become a standard procedure, the mechanistic details of the process are unknown. It has been suggested that endocytic uptake of lipoplexes is efficient, and transfectability is largely determined by later steps. In this article, we stained GM1-enriched membrane microdomains, a subclass of lipid rafts, with subunit B of cholera toxin and correlated transfection efficiency with their density by quantitatively evaluating microscopic images. We found a strong anticorrelation between the density of GM1-enriched membrane microdomains and the efficacy of transfection monitored by measuring the expression level of GFP in different cell lines transfected by lipofection using two different transfection agents. These findings imply that GM1-enriched membrane microdomains interfere with the process of lipofection. The blocked step must be endocytosis since the accumulation of fluorescently labeled plasmids was lower in cells with high content of GM1-enriched membrane microdomains. Such a correlation was not observed in cells transfected by electroporation. By comparing the efficiency of lipofection in several cell lines we found that those with a high density of GM1-enriched membrane microdomains were the most resistant to transfection. We conclude that the inhibition of lipofection by GM1-enriched membrane microdomains is a general rule, and that endocytosis of lipoplexes can be rate limiting in cells with high density of GM1-enriched membrane rafts. Copyright 2009 International Society for Advancement of Cytometry.

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

DEFF Research Database (Denmark)

Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

2017-01-01

, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

Acceleration of gene transfection efficiency in neuroblastoma cells through polyethyleneimine/poly(methyl methacrylate core-shell magnetic nanoparticles

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Tencomnao T

2012-06-01

Full Text Available Tewin Tencomnao,1,* Kewalin Klangthong,2,* Nuttaporn Pimpha,3 Saowaluk Chaleawlert-umpon,3 Somsak Saesoo,3 Noppawan Woramongkolchai,3 Nattika Saengkrit,31Center for Excellence in Omics-Nano Medical Technology Development Project, 2Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, 3National Nanotechnology Center, National Science and Technology Development Agency, Pathumthani, Thailand*Both authors contributed equally to this workBackground: 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

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

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Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

2017-02-01

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble.

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Kodama, Tetsuya; Tomita, Yukio; Koshiyama, Ken-Ichiro; Blomley, Martin J K

2006-06-01

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Science.gov (United States)

2011-01-01

Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease.

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Ye, Lei; Haider, Husnain Kh; Esa, Wahidah Bte; Su, Liping; Law, Peter K; Zhang, Wei; Lim, Yeanteng; Poh, Kian Keong; Sim, Eugene K W

2010-01-01

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

Photo-transfection of mammalian cells via femtosecond laser pulses

CSIR Research Space (South Africa)

Mthunzi, P

2009-06-01

Full Text Available on transient photo-transfection of ovary (CHO-Kl), neuroblastoma (NG-I08 & SKN-SH) and embryonic kidney (HEK-293) as well as primary non-differentiated stem cells (EI4g2a) using a tightly focused titanium sapphire laser beam (1.1 urn diameter spot size...

Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

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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

pSv3neo transfection and radiosensitivity of human cancer cell lines

International Nuclear Information System (INIS)

Parris, C.N.; Masters, J.R.W.; Green, M.H.L.

1990-01-01

Immortalisation of human fibroblasts by transfection with a plasmid, pSV3neo, results in an increase in their radioresistance. The change in radiosensitivity may either be a consequence of transformation or due to expression of the SV40 T-antigen in pSV3neo. To investigate these two possibilities, we transfected pSV3neo into cells already transformed and immortalised. The radiosensitivies of three human bladder cancer cell lines were unaltered in clones expressing T-antigen, indicating that the changes observed in fibroblasts probably are a consequence of transformation, and not the presence of SV40 T-antigen. (author)

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...... 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......, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine....

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

Directory of Open Access Journals (Sweden)

Wu K

2013-05-01

Full Text Available Kaimin Wu,1,* Jie Xu,2,* Mengyuan Liu,1 Wen Song,1 Jun Yan,1 Shan Gao,3 Lingzhou Zhao,2 Yumei Zhang1 1Department of Prosthetic Dentistry, 2Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 3The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China*Both authors contributed equally to this workAbstract: MicroRNA (miRNA regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall

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

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

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Lee K

2015-03-01

Full Text Available Kunwoo Lee,1,2 Pengzhi Yu,3 Nithya Lingampalli,1 Hyun Jin Kim,1 Richard Tang,1 Niren Murthy1,2 1Department of Bioengineering, University of California, Berkeley, CA, USA; 2UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA Abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from a-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. Keywords: direct cardiac

A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol-block-poly(disulfide histamine Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

Directory of Open Access Journals (Sweden)

Kangkang An

2014-05-01

Full Text Available A novel 4-arm poly(ethylene glycol-b-poly(disulfide histamine copolymer was synthesized by Michael addition reaction of poly(ethylene glycol (PEG vinyl sulfone and amine-capped poly(disulfide histamine oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter with almost neutral surface charge (+(5–10 mV. Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment, but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment. The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment. In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0. Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

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Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

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Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

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Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K; Champaneri, Shivam A; Taylor, Sarah; Davidson, Brian P; Zhao, Yan; Klibanov, Alexander L; Kuliszewski, Michael A; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R

2012-12-01

Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor.

Science.gov (United States)

Thumann, G; Stöcker, M; Maltusch, C; Salz, A K; Barth, S; Walter, P; Johnen, S

2010-02-01

Transplantation of pigment epithelial cells in patients with age-related macular degeneration and Parkinson's disease has the potential to improve functional rehabilitation. Genetic modification of cells before transplantation may allow the delivery of neuroprotective factors to achieve functional improvement. As transplantation of cells modified using viral vectors is complicated by the possible dissemination of viral particles and severe immune reactions, we have explored non-viral methods to insert genetic material in pigment epithelial cells. Using lipofection or nucleofection ARPE-19 cells, freshly isolated and primary retinal and iris pigment epithelial (IPE) cells were transfected with plasmids encoding green fluorescent protein (GFP) and with three plasmids encoding recombinant pigment epithelium-derived factor (PEDF) and GFP. Transfection efficiency was evaluated by fluorescence microscopy and stability of protein expression by immunoblotting. Pigment epithelial cells were successfully transfected with plasmid encoding GFP. Expression of GFP in ARPE-19 was transient, but was observed for up to 1 year in IPE cells. Analysis of pigment epithelial cells transfected with PEDF plasmids revealed that PEDF fusion proteins were successfully expressed and functionally active. In conclusion, efficient transfer of genetic information in pigment epithelial cells can be achieved using non-viral transfection protocols.

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

have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA......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...

Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.

Science.gov (United States)

Kuroda, Hitoshi; Kutner, Robert H; Bazan, Nicolas G; Reiser, Jakob

2009-05-01

During the past 12 years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.

[VEGF165 transfected endothelial progenitor cells mediated by lentivirus alleviated ALI in rats].

Science.gov (United States)

He, Zhaohui; He, Huiwei; Lu, Yuanhua; Chen, Zhi; Xu, Fanghua; Wang, Rongsheng; Yang, Chunli

2017-11-01

To investigate the protective effects of vascular endothelial growth factor-165 (VEGF165) transfected the endothelial progenitor cells (EPCs) mediated by lentivirus on acute lung injury (ALI) in rats. The mononuclear cells from the male Sprague-Dawley (SD) rats were isolated and cultured to get the EPCs for study. The lentivirus vector carrying the human VEGF165 gene was constructed. According to the random number table method, 90 male SD rats were divided into ALI model group, phosphate buffer solution (PBS) group, EPCs treatment group, none transfected EPCs treatment group and VEGF165 transfected EPCs treatment group, and the rats in each group were subdivided into 4, 12 and 48 hours subgroups, with 6 rats in each subgroup. The rat model of ALI was reproduced by intravenous injection of oleic acid (0.15 μL/g). Then each treatment group was given PBS, EPCs, none transfected EPCs and VEGF165 transfected EPCs respectively with the same volume of 0.2 mL. For the groups with cells, about 1×10 6 cells were contained. Abdominal aortic blood and lung tissue were harvested at 4, 12 and 48 hours. Arterial blood gas analysis was performed. The lung wet/dry weight ratio (W/D) was calculated. The expressions of induced nitric oxide synthase (iNOS), endothelin-1 (ET-1) and VEGF165 were determined by enzyme-linked immunosorbent assay (ELISA). After dyed with hematoxylin-eosin (HE), the lung tissue pathology was observed and the lung injury score was performed. Compared with the ALI model group, the arterial partial pressure of oxygen (PaO 2 ) in EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups was significantly increased from 4 hours, and lung W/D, expressions of iNOS and ET-1 were significantly decreased, and VEGF165 expression was significantly increased. Compared with the EPCs treatment group, the increase in PaO 2 , the decrease in lung W/D and expressions of iNOS and ET-1, and the increase in VEGF165 expression in VEGF165 transfected EPCs

Transfection of genetically encoded photoswitchable probes for STORM imaging.

Science.gov (United States)

Bates, Mark; Jones, Sara A; Zhuang, Xiaowei

2013-06-01

Conventional fluorescence microscopy is limited by its spatial resolution, leaving many biological structures too small to be studied in detail. Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. This protocol describes the transfection of genetically encoded photoswitchable probes for STORM imaging. It includes a discussion of how to choose a photoswitchable fluorescent protein; standard molecular biology techniques should be used to generate a plasmid containing the sequence of the photoswitchable protein linked to the gene of interest. Once the plasmid has been generated and has been verified, it can be introduced into cells via any standard means of gene delivery, such as lipofection or electroporation. Optimal conditions will vary considerably for different cell lines and plasmids. Here, we present an example protocol for the transfection of BS-C-1 cells with an mEos2-vimentin plasmid using the lipid-based reagent FuGENE6.

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.

Science.gov (United States)

Kneuer, C; Sameti, M; Bakowsky, U; Schiestel, T; Schirra, H; Schmidt, H; Lehr, C M

2000-01-01

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of

Glycosylphosphatidylinositol-anchored CD4 supports human immunodeficiency virus type 1 replication, but not cytopathic effect, in T-cell transfectants.

OpenAIRE

Marshall, W L; Mittler, E S; Avery, P; Lawrence, J P; Finberg, R W

1994-01-01

Despite equivalent p24 antigen production, HSB-2 T cells expressing glycosylphosphatidylinositol (GPi)-linked CD4 were productively infected without cell death or syncytium formation, unlike HSB-2 transfectants expressing wild-type CD4 (wtCD4). HSB-2 transfectants dually expressing wtCD4 and GPi-linked CD4 formed syncytia and died. Thus, wtCD4 expression is critical for human immunodeficiency virus cytopathic effect in HSB-2 transfectants.

Flow-through electroporation based on constant voltage for large-volume transfection of cells.

Science.gov (United States)

Geng, Tao; Zhan, Yihong; Wang, Hsiang-Yu; Witting, Scott R; Cornetta, Kenneth G; Lu, Chang

2010-05-21

Genetic modification of cells is a critical step involved in many cell therapy and gene therapy protocols. In these applications, cell samples of large volume (10(8)-10(9)cells) are often processed for transfection. This poses new challenges for current transfection methods and practices. Here we present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to approximately 20 mL/min) based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, we dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. We tested the delivery of pEFGP-C1 plasmids encoding enhanced green fluorescent protein into Chinese hamster ovary (CHO-K1) cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, approximately 75% of the viable CHO cells were transfected after the procedure. We also generalize the guidelines for scaling up these flow-through electroporation devices. We envision that this technique will serve as a generic and low-cost tool for a variety of clinical applications requiring large volume of transfected cells. Copyright 2010 Elsevier B.V. All rights reserved.

Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection

Science.gov (United States)

Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

2014-01-01

Abstract. In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage. PMID:25069006

Mutant p53 transfection of astrocytic cells results in altered cell cycle control, radiation sensitivity, and tumorigenicity

International Nuclear Information System (INIS)

Kanady, Kirk E.; Mei Su; Proulx, Gary; Malkin, David M.; Pardo, Francisco S.

1995-01-01

Introduction: Alterations in the p53 tumor suppressor gene are one of the most frequent genetic alterations in malignant gliomas. An understanding of the molecular genetic events leading to glial tumor progression would aid in designing therapeutic vectors for controlling these challenging tumor types. We investigated whether mutations in coding exons of the p53 gene result in functional changes altering cell cycle 'checkpoint' control and the intrinsic radiation sensitivity of glial cells. Methods: An astrocytic cell line was derived from a low grade astrocytoma and characterized to be of human karyotype and GFAP positivity. Additionally, the cellular population has never formed tumors in immune-deficient mice. At early passage ( 2 as parameters. Cell kinetic analyses after 2, 5, and 10 Gy of ionizing radiation were conducted using propidium iodide FACS analyses. Results: Overall levels of p53 expression were increased 5-10 fold in the transfected cellular populations. Astrocytic cellular populations transfected with mutant p53 revealed a statistically significant increase in levels of resistance to ionizing radiation in vitro (2-tailed test, SF2, MID). Astrocytic cellular populations transfected with mutant p53, unlike the parental cells, were tumorigenic in SCID mice. Cell kinetic analyses indicated that the untransfected cell line demonstrated dose dependent G1 and G2 arrests. Following transfection, however, the resultant cellular population demonstrated a predominant G2 arrest. Conclusions: Astrocytic cellular populations derived from low grade astrocytomas, are relatively radiation sensitive, non-tumorigenic, and have intact cell cycle ''checkpoints.'' Cellular populations resulting upon transfection of parental cells with a dominant negative p53 mutation, are relatively radiation resistant, when compared to both parental and mock-transfected cells. Transfected cells demonstrate abnormalities of cell cycle control at the G1/S checkpoint, increases in levels

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.

Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model

Directory of Open Access Journals (Sweden)

Hongchen Ji

2015-05-01

Full Text Available Background: The activation of tissue factor (TF is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF. Methods: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro. Results: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model. Conclusion: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.

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.

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.

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

Directory of Open Access Journals (Sweden)

Wang Y

2014-10-01

Full Text Available Yun Wang,1 Fu-xing Lin,2 Yu Zhao,1 Mo-zhen Wang,2 Xue-wu Ge,2 Zheng-xing Gong,1 Dan-dan Bao,1 Yu-fang Gu1 1Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, 2CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China Abstract: Novel submicron core-shell-structured chitosan-based composite particles ­encapsulated with enhanced green fluorescent protein plasmids (pEGFP were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC. pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. Keywords: gene therapy, gene transfection, hydroxybutyl chitosan, thiolated N-alkylated chitosan, pEGFP, complex coacervation

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 ...

Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-density transfection of suspension adapted HEK293S GnTI(-) cells

Czech Academy of Sciences Publication Activity Database

Bláha, J.; Pachl, Petr; Novák, Petr; Vaněk, O.

2015-01-01

Roč. 109, May (2015), s. 7-13 ISSN 1046-5928 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:OPPK(CZ) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388963 ; RVO:61388971 Keywords : LLT1 * HEK293S GnTI(-) * C-type lectin-like * NK cell * glycosylation * transfection Subject RIV: CE - Biochemistry Impact factor: 1.407, year: 2015

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.

MicroRNA-122 mimic transfection contributes to apoptosis in HepG2 cells.

Science.gov (United States)

Huang, Hongyan; Zhu, Yueyong; Li, Shaoyang

2015-11-01

There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer.

Science.gov (United States)

To, Kenneth K W; Poon, Daniel C; Wei, Yuming; Wang, Fang; Lin, Ge; Fu, Li-Wu

2016-06-01

We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer

Directory of Open Access Journals (Sweden)

Kenneth K.W. To

2016-06-01

Full Text Available We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1], can sensitize multidrug resistant (MDR colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]. This data article describes the possible circumvention of resistance to specifically platinum (Pt-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

Science.gov (United States)

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

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

Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging.

Science.gov (United States)

Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

2016-09-27

The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

Effects of Microbubble Size on Ultrasound-Mediated Gene Transfection in Auditory Cells

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Ai-Ho Liao

2014-01-01

Full Text Available Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-facilitated ultrasound (US technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

A versatile transfection assay system to evaluate the biological effects of diverse industrial chemicals.

Science.gov (United States)

Koizumi, Shinji; Ohno, Shotaro; Otsuka, Fuminori

2012-01-01

Gene expression processes are now recognized as important targets of the toxic effects exerted by industrial chemicals. The transient transfection assay is a powerful tool to evaluate such effects. Thus, we developed a versatile assay system by constructing a basic reporter plasmid in which the regulatory DNA sequence to be studied can easily be substituted. To verify the performance of this system, reporter plasmids carrying any of the three distinct regulatory sequences, estrogen responsive element (ERE), glucocorticoid responsive element (GRE) and xenobiotic responsive element (XRE) were constructed. After transfection of human cells, these plasmids successfully expressed the relevant reporter genes in response to specific inducers, β-estradiol, dexamethasone and 3-methylcholanthrene, respectively. Several industrial chemicals were assayed using these reporter plasmids, and the ability of p-dimethylaminoazobenzene to elevate GRE- and XRE-mediated transcription was detected. α-Naphthylamine and o-tolidine were also observed to increase the XRE-mediated response. The transfection assay system established here will be useful to evaluate the effects of a wide variety of industrial chemicals.

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

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Dawei Yu

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

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

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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.

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

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Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

2017-07-01

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

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.

Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

Science.gov (United States)

Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

2011-01-01

Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

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

Structure-transfection activity relationships in a series of novel cationic lipids with heterocyclic head-groups.

Science.gov (United States)

Ivanova, Ekaterina A; Maslov, Mikhail A; Kabilova, Tatyana O; Puchkov, Pavel A; Alekseeva, Anna S; Boldyrev, Ivan A; Vlassov, Valentin V; Serebrennikova, Galina A; Morozova, Nina G; Zenkova, Marina A

2013-11-07

Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.

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 d...

Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

Science.gov (United States)

Thobakgale, Lebogang; Manoto, Sello; Ombinda Lemboumba, Satuurnin; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.

Novel N,N '-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery--synthesis, in vitro transfection activity, and physicochemical characterization.

Science.gov (United States)

Spelios, Michael; Savva, Michalakis

2008-01-01

Novel N,N'-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/-4:1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/-4:1 and yielded increased lipofection activity at a lower charge ratio of +/-2:1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter>or=500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in-plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient amphiphiles, with gel-to-liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome-mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.

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

Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage

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Guan-gui Chen

2015-01-01

Full Text Available Polyethyleneimine-polyethylene glycol (PEI-PEG, a novel nanocarrier, has been used for transfection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Auditory brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

OpenAIRE

Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

1987-01-01

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and eff...

Fs-laser-induced Ca2+ concentration change during membrane perforation for cell transfection.

Science.gov (United States)

Baumgart, J; Bintig, W; Ngezahayo, A; Lubatschowski, H; Heisterkamp, A

2010-02-01

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.

Science.gov (United States)

Schomaker, Markus; Heinemann, Dag; Kalies, Stefan; Willenbrock, Saskia; Wagner, Siegfried; Nolte, Ingo; Ripken, Tammo; Murua Escobar, Hugo; Meyer, Heiko; Heisterkamp, Alexander

2015-02-03

In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat-shock-promoter-chloramphenicol-acetyltransferase construct.

Science.gov (United States)

Stiles, B; Heilmann, J; Sparks, R B; Santoso, A; Leopold, R A

1992-01-01

Expression of heat shock proteins (hsp) in the BRL-AG-3C cell line from the cotton boll weevil was examined. It was determined that the maximal expression of endogenous hsp occurred at 41 degrees C. Various transfection methods were then compared using this cell line in conjunction with a transiently expressed bacterial gene marker (chloramphenicol acetyltransferase) which was under the control of the Drosophila hsp 70 gene promoter. The cationic lipid preparation Lipofectin was found to be very efficient at transfecting the boll weevil cells. Polylysine and 20-hydroxyecdysone-conjugated polylysine were moderately effective, whereas polybrene and electroporation, under the conditions reported herein, were ineffective at transfecting this cell line.

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells.

Science.gov (United States)

Mennesson, Eric; Erbacher, Patrick; Piller, Véronique; Kieda, Claudine; Midoux, Patrick; Pichon, Chantal

2005-06-01

Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes. Copyright (c) 2005 John Wiley & Sons, Ltd.

A protocol for preparation and transfection of rat entorhinal cortex organotypic cultures for electrophysiological whole-cell recordings

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Nicholas I. Cilz

2017-01-01

Full Text Available Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of “bullets” carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.

Lipophosphoramidate-based bipolar amphiphiles: their syntheses and transfection properties.

Science.gov (United States)

Berchel, Mathieu; Le Gall, Tony; Lozach, Olivier; Haelters, Jean-Pierre; Montier, Tristan; Jaffrès, Paul-Alain

2016-03-14

Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity.

The effect of aloe emodin–encapsulated nanoliposome-mediated r-caspase-3 gene transfection and photodynamic therapy on human gastric cancer cells

International Nuclear Information System (INIS)

Li, Kai-Ting; Duan, Qin-Qin; Chen, Qing; He, Juan-Wen; Tian, Si; Lin, Hai-Dan; Gao, Qing; Bai, Ding-Qun

2015-01-01

Gastric carcinoma (GC) has high incidence and mortality rates in China. Surgery and chemotherapy are the main treatments. Photodynamic therapy (PDT) has become a new treatment modality, appearing in recent experimental studies and clinical trials in various tumors. This study explores the combined effect of gene transfection with PDT on GC cells using aloe emodin (AE)–encapsulated nanoliposomes, which acted as gene carrier as well as one photosensitizer (PS). AE-encapsulated nanoliposomes (nano-AE) were prepared by reverse evaporation method. Electron microscopy and nano-ZS90 analyzer were used to detect its morphology, size, and wavelength. Western blot was used to detect the expression of the caspase-3 after transfection. MTT assay and flow cytometry were employed to determine the cytotoxic and apoptotic rates, respectively. Hoechst 33342 staining was adopted to detect the morphological changes in death gastric cancer cells. Cellular reactive oxygen species (ROS) contents were measured by DCFH-DA staining. Outcomes demonstrated that the nano-AE has good properties as gene delivery carriers as well as a PS. The group in which the recombinant plasmid of r-caspase-3 was transfected had higher protein expression of the caspase-3 than controls, meanwhile the proliferation rates of the transfected cells were inhibited by the nano-AE-mediated PDT in an energy-dependent manner. In addition, in the transfected cells, the death rate increased to 77.3% as assessed 12 h after PDT (6.4 J/cm 2 ). Hochest 33342 staining also revealed that the death rate increased significantly in the transfected group compared with other groups. Compared to control groups, the production of ROS in nano-AE PDT group had quadrupled in SGC-7901 cells as early as 1 h after PDT, while it is similar to the group of nano-AE transfection and PDT. Nano-AE-mediated r-caspase-3 gene transfection coupled with PDT could inhibit the proliferation rate and increase the apoptotic rate remarkably in human

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

International Nuclear Information System (INIS)

Merkle, Thomas J.; O'Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H.

2004-01-01

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

Energy Technology Data Exchange (ETDEWEB)

Merkle, Thomas J.; O' Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H

2004-10-04

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.

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, competiti...

Structure-function relationships of new lipids designed for DNA transfection.

Science.gov (United States)

Dittrich, Matthias; Heinze, Martin; Wölk, Christian; Funari, Sergio S; Dobner, Bodo; Möhwald, Helmuth; Brezesinski, Gerald

2011-08-22

Cationic liposome/DNA complexes can be used as nonviral vectors for direct delivery of DNA-based biopharmaceuticals to damaged cells and tissues. To obtain more effective and safer liposome-based gene transfection systems, two cationic lipids with identical head groups but different chain structures are investigated with respect to their in vitro gene-transfer activity, their cell-damaging characteristics, and their physicochemical properties. The gene-transfer activities of the two lipids are very different. Differential scanning calorimetry and synchrotron small- and wide-angle X-ray scattering give valuable structural insight. A subgel-like structure with high packing density and high phase-transition temperature from gel to liquid-crystalline state are found for lipid 7 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2,N-bis(hexadecyl)propanediamide) containing two saturated chains. Additionally, an ordered head-group lattice based on formation of a hydrogen-bond network is present. In contrast, lipid 8 (N'-2-[(2,6-diamino-1-oxohexyl)amino]ethyl-2-hexadecyl-N-[(9Z)-octadec-9-enyl]propanediamide) with one unsaturated and one saturated chain shows a lower phase-transition temperature and a reduced packing density. These properties enhance incorporation of the helper lipid cholesterol needed for gene transfection. Both lipids, either pure or in mixtures with cholesterol, form lamellar phases, which are preserved after addition of DNA. However, the system separates into phases containing DNA and phases without DNA. On increasing the temperature, DNA is released and only a lipid phase without intercalated DNA strands is observed. The conversion temperatures are very different in the two systems studied. The important parameter seems to be the charge density of the lipid membranes, which is a result of different solubility of cholesterol in the two lipid membranes. Therefore, different binding affinities of the DNA to the lipid mixtures are achieved. Copyright © 2011

Adipogenic differentiation and EGFP gene transfection of amniotic fluid-derived stem cells from goat fetus at terminal gestational age.

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He, Xiao-Ying; Zheng, Yue-Mao; Qiu, Shuang; Qi, Ying-Pei; Zhang, Yong

2011-08-01

The aims of this study were to determine whether stem cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and to determine if these stem cells could differentiate into adipogenic cells and be transfected with a reporter gene, EGFP (enhanced green fluorescent protein). The stem cells were isolated from amniotic fluid of goat fetus at terminal gestational age, induced to differentiate into adipogenic cells in vitro and transfected with the EGFP gene using lipofection. Markers associated with undifferentiated AFS (amniotic fluid-derived stem) cells were tested by RT (reverse transcription)-PCR. The results demonstrated that AFS cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and could differentiate into adipogenic cells. The EGFP gene was transfected into AFS cells successfully. EGFP gene transfection efficiency of the three groups of transgenic AFS cells were 26.0, 29.9 and 30.5%, respectively. Both transgenic and wild-type AFS cells could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4) and Nanog.

Factors influencing transfection efficiency of pIDUA/nanoemulsion complexes in a mucopolysaccharidosis type I murine model

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Fraga M

2017-03-01

Full Text Available Michelle Fraga,1,2 Talita Giacomet de Carvalho,2,3 Juliana Bidone,1 Roselena Silvestri Schuh,1,2 Ursula Matte,2,3 Helder Ferreira Teixeira1 1Pharmaceutical Sciences Graduate Program, Universidade Federal do Rio Grande do Sul, 2Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, 3Genetics and Molecular Biology Graduate Program, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Abstract: Mucopolysaccharidosis type I (MPS I is an autosomal disease caused by alpha-L-iduronidase (IDUA deficiency. This study used IDUA knockout mice as a model to evaluate whether parameters such as dose of plasmid and time of treatment could influence the transfection efficiency of complexes formed with PEGylated cationic nanoemulsions and plasmid (pIDUA, which contains the gene that encodes for IDUA. Formulations were composed of medium chain triglycerides, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino[polyethylene glycol]-2000, 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP, glycerol, and water and were prepared by the adsorption or encapsulation of preformed pIDUA–DOTAP complexes by high-pressure homogenization. A progressive increase in IDUA expression was observed with an increase in the dose and time of transfection for mice treated with both complexes (adsorbed and encapsulated, especially in the liver. Regardless of the complex administered, a significant increase in IDUA activity was detected in lungs and liver compared with nontreated MPS I when a dose of 60 µg was administered and IDUA activity was measured 7 days postadministration. Tissue sections of major organs showed no presence of cell necrosis, inflammatory infiltrate, or an increase in apoptosis. Furthermore, immunohistochemistry for CD68 showed no difference in the number of macrophage cells in treated and nontreated animals, indicating the absence of inflammatory reaction

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

Science.gov (United States)

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

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Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

2018-04-02

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

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.

Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts

International Nuclear Information System (INIS)

Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.

1987-01-01

Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA

Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

Directory of Open Access Journals (Sweden)

Akkina Ramesh

2008-07-01

Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

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...

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

Science.gov (United States)

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

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...... 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....... 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...

Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

Science.gov (United States)

Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

2016-12-30

This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

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....

Biodegradable gadolinium-chelated cationic poly(urethane amide) copolymers for gene transfection and magnetic resonance imaging

Energy Technology Data Exchange (ETDEWEB)

Gao, Xiaolong [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China); Wang, Gangmin [Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040 (China); Shi, Ting [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Shao, Zhihong [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China); Zhao, Peng; Shi, Donglu [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Ren, Jie [Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji University, 4800 Caoan Road, Shanghai 201804 (China); Lin, Chao, E-mail: chaolin@tongji.edu.cn [The Institute for Translational Nanomedicine, Shanghai East Hospital, Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Peijun, E-mail: tjpjwang@sina.com [Department of Radiology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065 (China)

2016-08-01

Theranostic nano-polyplexes containing gene and imaging agents hold a great promise for tumor diagnosis and therapy. In this work, we develop a group of new gadolinium (Gd)-chelated cationic poly(urethane amide)s for gene delivery and T{sub 1}-weighted magnetic resonance (MR) imaging. Cationic poly(urethane amide)s (denoted as CPUAs) having multiple disulfide bonds, urethane and amide linkages were synthesized by stepwise polycondensation reaction between 1,4-bis(3-aminopropyl)piperazine and a mixture of di(4-nitrophenyl)-2, 2′-dithiodiethanocarbonate (DTDE-PNC) and diethylenetriaminepentaacetic acid (DTPA) dianhydride at varied molar ratios. Then, Gd-chelated CPUAs (denoted as GdCPUAs) were produced by chelating Gd(III) ions with DTPA residues of CPUAs. These GdCPUAs could condense gene into nanosized and positively-charged polyplexes in a physiological condition and, however, liberated gene in an intracellular reductive environment. In vitro transfection experiments revealed that the GdCPUA at a DTDE-PNC/DTPA residue molar ratio of 85/15 induced the highest transfection efficiency in different cancer cells. This efficiency was higher than that yielded with 25 kDa branched polyethylenimine as a positive control. GdCPUAs and their polyplexes exhibited low cytotoxicity when an optimal transfection activity was detected. Moreover, GdCPUAs may serve as contrast agents for T{sub 1}-weighted magnetic resonance imaging. The results of this work indicate that biodegradable Gd-chelated cationic poly(urethane amide) copolymers have high potential for tumor theranostics. - Highlights: • Novel cationic gadolinium-chelated poly(urethane amide)s (GdCPUAs) are prepared. • GdCPUAs can induce a high transfection efficacy in different cancer cells. • GdCPUAs reveal good cyto-compatibility against cancer cells. • GdCPUAs may be applied as T{sub 1}-contrast agents for magnetic resonance imaging. • GdCPUAs hold high potential for cancer theranostics.

Biodegradable gadolinium-chelated cationic poly(urethane amide) copolymers for gene transfection and magnetic resonance imaging

International Nuclear Information System (INIS)

Gao, Xiaolong; Wang, Gangmin; Shi, Ting; Shao, Zhihong; Zhao, Peng; Shi, Donglu; Ren, Jie; Lin, Chao; Wang, Peijun

2016-01-01

Theranostic nano-polyplexes containing gene and imaging agents hold a great promise for tumor diagnosis and therapy. In this work, we develop a group of new gadolinium (Gd)-chelated cationic poly(urethane amide)s for gene delivery and T 1 -weighted magnetic resonance (MR) imaging. Cationic poly(urethane amide)s (denoted as CPUAs) having multiple disulfide bonds, urethane and amide linkages were synthesized by stepwise polycondensation reaction between 1,4-bis(3-aminopropyl)piperazine and a mixture of di(4-nitrophenyl)-2, 2′-dithiodiethanocarbonate (DTDE-PNC) and diethylenetriaminepentaacetic acid (DTPA) dianhydride at varied molar ratios. Then, Gd-chelated CPUAs (denoted as GdCPUAs) were produced by chelating Gd(III) ions with DTPA residues of CPUAs. These GdCPUAs could condense gene into nanosized and positively-charged polyplexes in a physiological condition and, however, liberated gene in an intracellular reductive environment. In vitro transfection experiments revealed that the GdCPUA at a DTDE-PNC/DTPA residue molar ratio of 85/15 induced the highest transfection efficiency in different cancer cells. This efficiency was higher than that yielded with 25 kDa branched polyethylenimine as a positive control. GdCPUAs and their polyplexes exhibited low cytotoxicity when an optimal transfection activity was detected. Moreover, GdCPUAs may serve as contrast agents for T 1 -weighted magnetic resonance imaging. The results of this work indicate that biodegradable Gd-chelated cationic poly(urethane amide) copolymers have high potential for tumor theranostics. - Highlights: • Novel cationic gadolinium-chelated poly(urethane amide)s (GdCPUAs) are prepared. • GdCPUAs can induce a high transfection efficacy in different cancer cells. • GdCPUAs reveal good cyto-compatibility against cancer cells. • GdCPUAs may be applied as T 1 -contrast agents for magnetic resonance imaging. • GdCPUAs hold high potential for cancer theranostics.

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

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

Energy Technology Data Exchange (ETDEWEB)

Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

International Nuclear Information System (INIS)

Su, L.-N.; Little, J.B.

1992-01-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author)

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.

Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

International Nuclear Information System (INIS)

Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

1995-01-01

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 α-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 pCMVΒ vector; and (2) the antibiotic hygromycin-resistant transfected cells

Human Breast Adipose-Derived Stem Cells Transfected with the Stromal Cell-Derived Factor-1 Receptor CXCR4 Exhibit Enhanced Viability in Human Autologous Free Fat Grafts

Directory of Open Access Journals (Sweden)

Fang-tian Xu

2014-11-01

analysis revealed that both types of HBASCs-treated transplants consisted predominantly of adipose tissue, unlike the control transplants, and also presented significantly less fat necrosis and fibrosis. The CXCR4-transfected HBASCs-treated transplants had a significantly higher capillary density than did the other transplants and showed GFP and CD31 double-positive cells (i.e., ASCs-derived endothelial cells. The mRNA expression of CXCR4 and SDF-1α was much higher in the CXCR4-transfected HBASCs transplants than in the other three transplants. Conclusions: Our data demonstrated that HBASCs can enhance the survival and quality of transplanted free fat tissues. Moreover, CXCR4 transfection of these HBASCs could augment this effect. Stimulation of angiogenesis and decreased fat cell apoptosis due to the recruitment of endothelial progenitor cells (EPCs and an increase in graft revascularization are potential mechanisms underlying the improved long-term survival of free fat transplants following CXCR4-transfected HBASCs treatment.

In Vitro Repair of UV-Irradiated Micrococcus luteus Bacteriophage N1 Transfecting DNA 1

Science.gov (United States)

Mahler, Inga; George, Jeanne; Grossman, Lawrence

1974-01-01

Calcium-treated UV-sensitive, host cell reactivation− strains of Micrococcus luteus are infected with UV-irradiated N1 DNA. In strains lacking UV endonuclease, in vitro treatment of the irradiated DNA results in transfection enhancement. PMID:4823319

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

Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (qp)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), Li......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...... of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 m...

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene

International Nuclear Information System (INIS)

Li, G.C.; Li, Ligeng; Liu, Yunkang; Mak, J.Y.; Chen, Lili; Lee, W.M.F.

1991-01-01

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. The authors have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, low-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45C and their thermal survivals were determined by colony-formation assay, they found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress

Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

Science.gov (United States)

Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

2015-04-01

Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

Optimal transfection methods and comparison of PK-15 and Dulac cells for rescue of chimeric porcine circovirus type 1-2.

Science.gov (United States)

Li, Jizong; Yu, Tianqi; Zhou, Jinzhu; Tu, Wei; Gao, Song; Liu, Xiufan

2014-11-01

A chimeric porcine circovirus type 1-2 (PCV1-2) infectious DNA clone has low transfection efficiency and exhibits low levels of proliferation. Electroporation and lipofection parameters were optimized for PK-15 and Dulac cells with the purpose of increasing the efficiency for rescuing infectious PCV1-2. Titers of PCV1-2 in Dulac cells were 100-fold higher than those in PK-15 cells following transfection. The electroporation efficiency into Dulac cells was high when three 400 μs pulses at 250 V with 6 μg of plasmid DNA was used, lipofection efficiency was high when the ratio of DNA to transfection reagent was 1:3. The proportion of infected cells was 55.6% compared with 44.2%, for the electroporation and lipofection techniques respectively. Virus titers were higher in Dulac cells, from 10(4.44) to 10(5.32)TCID50/mL compared with 10(1.90)-10(3.38)TCID(50)/mL for PK-15 cells. Dulac cells were more permissive to PCV1-2 than PK-15 cells regardless of the transfection technique. Copyright © 2014 Elsevier B.V. All rights reserved.

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)

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

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.

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

KAUST Repository

Deng, Peigang; Chang, Donald C.; Lee, Yi Kuen; Zhou, Junwei; Li, Gang

2011-01-01

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.

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.

Establishment of Lipofection Protocol for Efficient miR-21 Transfection into Cortical Neurons In Vitro.

Science.gov (United States)

Han, Zhaoli; Ge, Xintong; Tan, Jin; Chen, Fanglian; Gao, Huabin; Lei, Ping; Zhang, Jianning

2015-12-01

Dysregulated microRNAs in neurons could cause many nervous system diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNA modulators targeting neurons with minimal off-target effects. The study aimed to establish a lipofection protocol to upregulate expression levels of miR-21 in neurons under different conditions, including different serum-free medium, transfection conditions, and reagent concentration, by evaluating the expression levels of miR-21 and neuron injury. The expression levels of miR-21 were higher in neurons transfected by Neurobasal-A than by DMEM. Expression levels of miR-21 were already the highest at the ratio RNAiMAX:miR-21 = 3:5, but the increase of RNAiMAX's concentration had not caused the further upregulation of expression level of miR-21. Neuron injury was condition dependent and dose dependent after transfection. Compared to S-Neurobasal groups, neurons have a smaller injury in N-Neurobasal groups, and compared to ratios RNAiMAX:miR-21 = 4:5, 5:5, neuron injury was smaller at ratios of RNAiMAX:miR-21 = 1:5, 2:5, 3:5. Without the pretreatment of starvation in vitro, the lipofection protocol was that RNAiMAX/miR-21 agomir complexes were diluted in Neurobasal-A at the ratio of RNAiMAX:miR-21 = 3:5.

Transfection of embryonated Muscovy duck eggs with a recombinant plasmid is suitable for rescue of infectious Muscovy duck parvovirus.

Science.gov (United States)

Wang, Jianye; Huang, Yu; Ling, Jueyi; Wang, Zhixiang; Zhu, Guoqiang

2017-12-01

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

Science.gov (United States)

Sharifi Tabar, Mehdi; Hesaraki, Mahdi; Esfandiari, Fereshteh; Sahraneshin Samani, Fazel; Vakilian, Haghighat; Baharvand, Hossein

2015-01-01

Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene. Results Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 μg and the density of the sub- jected cells (5×105and 1×106cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

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

with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75 % with survival rates of >90 %. This chapter describes...

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

Physically and chemically stable ionic liquid-infused textured surfaces showing excellent dynamic omniphobicity

Energy Technology Data Exchange (ETDEWEB)

Miranda, Daniel F.; Urata, Chihiro; Masheder, Benjamin; Dunderdale, Gary J.; Hozumi, Atsushi, E-mail: a.hozumi@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98, Anagahora, Shimo-Shidami, Moriyama-ku, Nagoya, Aichi 463-8560 (Japan); Yagihashi, Makoto [Nagoya Municipal Industrial Research Institute, Rokuban, Atsuta-ku, Nagoya 456-0058 (Japan)

2014-05-01

A fluorinated and hydrophobic ionic liquid (IL), 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, effectively served as an advantageous lubricating liquid for the preparation of physically and chemically stable omniphobic surfaces based on slippery liquid-infused porous surfaces. Here, we used particulate microstructures as supports, prepared by the chemical vapor deposition of 1,3,5,7-tetramethylcyclotetrasiloxane and subsequent surface modification with (3-aminopropyl)triethoxysilane. Confirmed by SEM and contact angle measurements, the resulting IL-infused microtextured surfaces are smooth and not only water but also various low surface tension liquids can easily slide off at low substrate tilt angles of <5°, even after exposure to high temperature, vacuum, and UV irradiation.

Physically and chemically stable ionic liquid-infused textured surfaces showing excellent dynamic omniphobicity

Directory of Open Access Journals (Sweden)

Daniel F. Miranda

2014-05-01

Full Text Available A fluorinated and hydrophobic ionic liquid (IL, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl imide, effectively served as an advantageous lubricating liquid for the preparation of physically and chemically stable omniphobic surfaces based on slippery liquid-infused porous surfaces. Here, we used particulate microstructures as supports, prepared by the chemical vapor deposition of 1,3,5,7-tetramethylcyclotetrasiloxane and subsequent surface modification with (3-aminopropyltriethoxysilane. Confirmed by SEM and contact angle measurements, the resulting IL-infused microtextured surfaces are smooth and not only water but also various low surface tension liquids can easily slide off at low substrate tilt angles of <5°, even after exposure to high temperature, vacuum, and UV irradiation.

Population Games, Stable Games, and Passivity

Directory of Open Access Journals (Sweden)

Michael J. Fox

2013-10-01

Full Text Available The class of “stable games”, introduced by Hofbauer and Sandholm in 2009, has the attractive property of admitting global convergence to equilibria under many evolutionary dynamics. We show that stable games can be identified as a special case of the feedback-system-theoretic notion of a “passive” dynamical system. Motivated by this observation, we develop a notion of passivity for evolutionary dynamics that complements the definition of the class of stable games. Since interconnections of passive dynamical systems exhibit stable behavior, we can make conclusions about passive evolutionary dynamics coupled with stable games. We show how established evolutionary dynamics qualify as passive dynamical systems. Moreover, we exploit the flexibility of the definition of passive dynamical systems to analyze generalizations of stable games and evolutionary dynamics that include forecasting heuristics as well as certain games with memory.

Transfection and imaging of diamond nanocrystals as scattering optical labels

International Nuclear Information System (INIS)

Smith, Bradley R.; Niebert, Marcus; Plakhotnik, Taras; Zvyagin, Andrei V.

2007-01-01

We report on the first demonstration of nanodiamond (ND) as a scattering optical label in a biological environment. NDs were efficiently transfected into cells using cationic liposomes, and imaged using differential interference and Hoffman modulation 'space' contrast microscopy techniques. We have shown that 55 nm NDs are biologically inert and produce a bright signal compared to the cell background. ND as a scattering label presents the possibility for extended biological imaging with relatively little thermal or biochemical perturbations due to the optical transparency and biologically inert nature of diamond

Stable rotating dipole solitons in nonlocal media

DEFF Research Database (Denmark)

Lopez-Aguayo, Servando; Skupin, Stefan; Desyatnikov, Anton S.

2006-01-01

We present the first example of stable rotating two-soliton bound states in nonlinear optical media with nonlocal response. We show that, in contrast to media with local response, nonlocality opens possibilities to generate stable azimuthons.......We present the first example of stable rotating two-soliton bound states in nonlinear optical media with nonlocal response. We show that, in contrast to media with local response, nonlocality opens possibilities to generate stable azimuthons....

Conceptual and technical aspects of transfection and gene delivery.

Science.gov (United States)

Kaestner, Lars; Scholz, Anke; Lipp, Peter

2015-03-15

Genetically modified animals are state of the art in biomedical research as gene therapy is a promising perspective in the attempt to cure hereditary diseases. Both approaches have in common that modified or corrected genetic information must be transferred into cells in general or into particular cell types of an organism. Here we give an overview of established and emerging methods of transfection and gene delivery and provide conceptual and technical advantages and drawbacks of their particular use. Additionally, based on a flow chart, we compiled a rough guideline to choose a gene transfer method for a particular field of application. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS.

Science.gov (United States)

Brown, C A; Charlton, S J; Boarder, M R

1997-03-01

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2',4' disulphonic acid (PPADS) act as antagonists at transfected P2Y1 receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors. 2. The expression of either P2Y1 or P2Y2 receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [3H]-inositol(poly)phosphates([3H]-InsPx) compared to cells not expressing these receptors. This elevation is much greater in P2Y1 transfectants than in P2Y, transfectants. 3. Both PPADS and suramin reduced this basal level of [3H]-InsPx accumulation in the P2Y1 expressing cells. 4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5'-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist. 5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [3H]-InsPx accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y1 expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

Science.gov (United States)

Lawley, Blair; Munro, Karen; Sims, Ian M.; Lee, Julian; Butts, Christine A.; Roy, Nicole

2014-01-01

Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect 13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the 13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism. PMID:24487527

Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

Science.gov (United States)

Parvizi, Paria; Jubeli, Emile; Raju, Liji; Khalique, Nada Abdul; Almeer, Ahmed; Allam, Hebatalla; Manaa, Maryem Al; Larsen, Helge; Nicholson, David; Pungente, Michael D; Fyles, Thomas M

2014-01-30

This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

mRNA transfection of mouse and human neural stem cell cultures

OpenAIRE

McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

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JIANG Hua-wei

2014-09-01

Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed ＞90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo

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Krista Freimann

2018-03-01

Full Text Available Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

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

Energy Technology Data Exchange (ETDEWEB)

Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F. (Hagedorn Research Laboratory, Gentofte (Denmark))

1988-09-01

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.

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

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.

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

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Ikeda, Ritsuko; Kurokawa, Manae S; Chiba, Shunmei; Yoshikawa, Hideshi; Hashimoto, Takuo; Tadokoro, Mamoru; Suzuki, Noboru

2004-10-01

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

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.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

Science.gov (United States)

Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

2017-08-10

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles.

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Juan Ramón Vanegas Sáenz

Full Text Available 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.

Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

Science.gov (United States)

Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

2006-08-01

Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

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......RNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate...... 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....

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

Science.gov (United States)

Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki

2017-08-01

Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration. Copyright © 2017 John Wiley & Sons, Ltd.

Efficient transfection of Xenobiotic Responsive Element-biosensor plasmid using diether lipid and phosphatidylcholine liposomes in differentiated HepaRG cells.

Science.gov (United States)

Demazeau, Maxime; Quesnot, Nicolas; Ripoche, Nicolas; Rauch, Claudine; Jeftić, Jelena; Morel, Fabrice; Gauffre, Fabienne; Benvegnu, Thierry; Loyer, Pascal

2017-05-30

In this study, we evaluated cationic liposomes prepared from diether-NH 2 and egg phosphatidylcholine (EPC) for in vitro gene delivery. The impact of the lipid composition, i.e. the EPC and Diether-NH 2 molar ratio, on in vitro transfection efficiency and cytotoxicity was investigated using the human HEK293T and hepatoma HepaRG cells known to be permissive and poorly permissive cells for liposome-mediated gene transfer, respectively. Here, we report that EPC/Diether-NH 2 -based liposomes enabled a very efficient transfection with low cytotoxicity compared to commercial transfection reagents in both HEK293T and proliferating progenitor HepaRG cells. Taking advantage of these non-toxic EPC/Diether-NH 2 -based liposomes, we developed a method to efficiently transfect differentiated hepatocyte-like HepaRG cells and a biosensor plasmid containing a Xenobiotic Responsive Element and a minimal promoter driving the transcription of the luciferase reporter gene. We demonstrated that the luciferase activity was induced by a canonical inducer of cytochrome P450 genes, the benzo[a]pyrene, and two environmental contaminants, the fluoranthene, a polycyclic aromatic hydrocarbon, and the endosulfan, an organochlorine insecticide, known to induce toxicity and genotoxicity in differentiated HepaRG cells. In conclusion, we established a new efficient lipofection-mediated gene transfer in hepatocyte-like HepaRG cells opening new perspectives in drug evaluation relying on xenobiotic inducible biosensor plasmids. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

Science.gov (United States)

Lang, Annemarie; Neuhaus, Johannes; Pfeiffenberger, Moritz; Schröder, Erik; Ponomarev, Igor; Weber, Yvonne; Gaber, Timo; Schmidt, Michael F G

2014-01-01

Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.

High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

DEFF Research Database (Denmark)

Adler, Andrew F; Speidel, Alessondra T; Christoforou, Nicolas

2011-01-01

of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM....... Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library...... and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems....

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

Energy Technology Data Exchange (ETDEWEB)

Wu Xuewen; Ding Dalian [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Jiang Haiyan [State University of New York at Buffalo, Center for Hearing and Deafness (United States); XingXiaowei [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Huang, Suping [Central South University, State Key Laboratory of Powder Metallurgy (China); Liu Hong [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Chen Zhedong [Central South University, State Key Laboratory of Powder Metallurgy (China); Sun Hong, E-mail: shjhaj@vip.163.com [Central South University, Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital (China)

2012-01-15

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 {+-} 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of

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

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Fu-qing Zhong

2014-01-01

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

Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

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Tripti Tamhane

2015-12-01

Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

Nonbilayer phase of lipoplex-membrane mixture determines endosomal escape of genetic cargo and transfection efficiency

NARCIS (Netherlands)

Zuhorn, IS; Bakowsky, U; Polushkin, E; Visser, WH; Stuart, MCA; Engberts, JBFN; Hoekstra, D; Visser, Willy H.

Cationic lipids are widely used for gene delivery, and inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid in cationic lipid-DNA formulations often promotes transfection efficacy. To investigate the significance of DOPE's preference to adopt a hexagonal phase in the mechanism of

IL-1RA gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules could alleviate rheumatoid arthritis.

Science.gov (United States)

Hu, Jianhua; Li, Hongjian; Chi, Guanhao; Yang, Zhao; Zhao, Yi; Liu, Wei; Zhang, Chao

2015-01-01

In order to investigate the encapsulation of interleukin 1 receptor antagonist (IL-RA) gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of interleukin 1 receptor antagonist (IL-RA) to treat Rheumatoid arthritis (RA). We transfect mesenchymal stem cells with IL-RA gene, and quantify the IL-RA proteins released from the encapsulated cells followed by microencapsulation of recombinant mesenchymal stem cells, and thus observe the permeability of APA microcapsules and evaluate clinical effects after induction and treatment of collagen-induced arthritis (CIA). The concentration of IL-RA in the supernatant was determined by IL-RA ELISA kit by run in technical triplicates using samples from three separate mice. Encapsulated IL-RA gene-transfected cells were capable of constitutive delivery of IL-RA proteins for at least 30 days. Moreover, the APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Also, it has been found that the APA microcapsules can significantly attenuate collagen induced arthritis after delivering of APA microcapsules to rats. Our results demonstrated that the nonautologous IL-RA gene-transfected stem cells are of potential utility for RA therapy.

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

RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

International Nuclear Information System (INIS)

Kleinschmidt, A.M.; Pederson, T.

1990-01-01

The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

Remarks on stable and quasi-stable k-strings at large N

International Nuclear Information System (INIS)

Armoni, A.; Shifman, M.

2003-01-01

We discuss k-strings in the large-N Yang-Mills theory and its supersymmetric extension. Whereas the tension of the bona fide (stable) QCD string is expected to depend only on the N-ality of the representation, tensions that depend on specific representation R are often reported in the lattice literature. In particular, adjoint strings are discussed and found in certain simulations. We clarify this issue by systematically exploiting the notion of the quasi-stable strings which becomes well-defined at large N. The quasi-stable strings with representation-dependent tensions decay, but the decay rate (per unit length per unit time) is suppressed as Λ 2 F(N) where F(N) falls off as a function of N. It can be determined on the case-by-case basis. The quasi-stable strings eventually decay into stable strings whose tension indeed depends only on the N-ality. We also briefly review large-N arguments showing why the Casimir formula for the string tension cannot be correct, and present additional arguments in favor of the sine formula. Finally, we comment on the relevance of our estimates to Euclidean lattice measurements

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...

Lipid chain geometry of C14 glycerol-based lipids: effect on lipoplex structure and transfection.

Science.gov (United States)

Kudsiova, Laila; Ho, Jimmy; Fridrich, Barbara; Harvey, Richard; Keppler, Melanie; Ng, Tony; Hart, Stephen L; Tabor, Alethea B; Hailes, Helen C; Lawrence, M Jayne

2011-02-01

The effects have been determined of a systematic alteration of the alkyl chain geometry of a C14 analogue of DOTMA on the detailed molecular architecture of the resulting cationic vesicles formed both in the absence and presence of 50 mol% DOPE, and of the lipoplexes prepared from these vesicles using either calf thymus or plasmid DNA. The C14 DOTMA analogues studied involved cis- or trans-double bonds at positions Δ9 or Δ11, and a compound (ALK) featuring an alkyne at position C9. For all of these analogues, examination by light scattering and neutron scattering, zeta potential measurement, and negative staining electron microscopy showed that there were no significant differences in the structures or charges of the vesicles or of the resulting lipoplexes, regardless of the nature of the DNA incorporated. Differences were observed, however, between the complexes formed by the various lipids when examining the extent of complexation and release by gel electrophoresis, where the E-lipids appeared to complex the DNA more efficiently than all other lipids tested. Moreover, the lipoplexes prepared from the E-lipids were the most effective in transfection of MDA-MB-231 breast cancer cells. As indicated through confocal microscopy studies, the E-lipids also showed a higher internalisation capacity and a more diffuse cellular distribution, possibly indicating a greater degree of endosomal escape and/or nuclear import. These observations suggest that the extent of complexation is the most important factor in determining the transfection efficiency of the complexes tested. At present it is unclear why the E-lipids were more effective at complexing DNA, although it is thought that the effective area per molecule occupied by the cationic lipid and DOPE head groups, and therefore the density of positive charges on the surface of the bilayer most closely matches the negative charge density of the DNA molecule. From a consideration of the geometry of the cationic lipids it is

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.

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

Science.gov (United States)

Kofron, Michelle D; Laurencin, Cato T

2004-06-01

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

Biophysical and lipofection studies of DOTAP analogs.

Science.gov (United States)

Regelin, A E; Fankhaenel, S; Gürtesch, L; Prinz, C; von Kiedrowski, G; Massing, U

2000-03-15

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.

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

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

International Nuclear Information System (INIS)

Wu Xuewen; Ding Dalian; Jiang Haiyan; XingXiaowei; Huang, Suping; Liu Hong; Chen Zhedong; Sun Hong

2012-01-01

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for

MicroRNA transfection and AGO-bound CLIP-seq data sets reveal distinct determinants of miRNA action

DEFF Research Database (Denmark)

Wen, Jiayu; Parker, Brian J; Jacobsen, Anders

2011-01-01

the predictive effect of target flanking features. We observe distinct target determinants between expression-based and CLIP-based data. Target flanking features such as flanking region conservation are an important AGO-binding determinant-we hypothesize that CLIP experiments have a preference for strongly bound......Microarray expression analyses following miRNA transfection/inhibition and, more recently, Argonaute cross-linked immunoprecipitation (CLIP)-seq assays have been used to detect miRNA target sites. CLIP and expression approaches measure differing stages of miRNA functioning-initial binding of the mi...... miRNP-target interactions involving adjacent RNA-binding proteins that increase the strength of cross-linking. In contrast, seed-related features are major determinants in expression-based studies, but less so for CLIP-seq studies, and increased miRNA concentrations typical of transfection studies...

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

Science.gov (United States)

Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

2003-07-15

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

International Nuclear Information System (INIS)

Kapp, L.N.; Painter, R.B.

1989-01-01

SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

International Nuclear Information System (INIS)

Duffy, J.J.; Carper, S.W.; Gerner, E.W.

1987-01-01

Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

The Myopic Stable Set for Social Environments

NARCIS (Netherlands)

Demuynck, Thomas; Herings, P. Jean-Jacques; Saulle, Riccardo; Seel, Christian

2017-01-01

We introduce a new solution concept for models of coalition formation, called the myopic stable set. The myopic stable set is defined for a very general class of social environments and allows for an infinite state space. We show that the myopic stable set exists and is non-empty. Under minor

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

Science.gov (United States)

Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

2014-01-15

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Stable configurations in social networks

Science.gov (United States)

Bronski, Jared C.; DeVille, Lee; Ferguson, Timothy; Livesay, Michael

2018-06-01

We present and analyze a model of opinion formation on an arbitrary network whose dynamics comes from a global energy function. We study the global and local minimizers of this energy, which we call stable opinion configurations, and describe the global minimizers under certain assumptions on the friendship graph. We show a surprising result that the number of stable configurations is not necessarily monotone in the strength of connection in the social network, i.e. the model sometimes supports more stable configurations when the interpersonal connections are made stronger.

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

OpenAIRE

Rosenberg, Z F; Sahagan, B G; Snyder, H W; Worley, M B; Essex, M; Haseltine, W A

1981-01-01

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells express...

An Atypical Human Induced Pluripotent Stem Cell Line With a Complex, Stable, and Balanced Genomic Rearrangement Including a Large De Novo 1q Uniparental Disomy

Science.gov (United States)

Steichen, Clara; Maluenda, Jérôme; Tosca, Lucie; Luce, Eléanor; Pineau, Dominique; Dianat, Noushin; Hannoun, Zara; Tachdjian, Gérard; Melki, Judith

2015-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts. PMID:25650439

The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

International Nuclear Information System (INIS)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

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.

Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

Science.gov (United States)

Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

2016-08-01

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

DEFF Research Database (Denmark)

Rasmussen, A B; Zocca, M-B; Bonefeld, C M

2004-01-01

Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent...... on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...... to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell...

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

Science.gov (United States)

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

MicroRNA-mediated myostatin silencing in caprine fetal fibroblasts.

Directory of Open Access Journals (Sweden)

Bushuai Zhong

Full Text Available Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi mediated by microRNAs (miRNAs is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN response genes, such as interferon beta (IFN-β and 2'-5'-oligoadenylate synthetase 2 (OAS2, were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats.

Guanidinylated polyethyleneimine-polyoxypropylene-polyoxyethylene conjugates as gene transfection agents.

Science.gov (United States)

Bromberg, Lev; Raduyk, Svetlana; Hatton, T Alan; Concheiro, Angel; Rodriguez-Valencia, Cosme; Silva, Maite; Alvarez-Lorenzo, Carmen

2009-05-20

Conjugates of linear and branched polyethyleneimine (PEI) and monoamine polyether Jeffamine M-2070 (PO/EO mol ratio 10/31, 2000 Da) were synthesized through polyether activation by cyanuric chloride followed by attachment to PEI and guanidinylation by 1H-pyrazole-carboxamidine hydrochloride. The resulting guanidinylated PEI-polyether conjugates (termed gPEI-Jeffamine) efficiently complexed plasmid DNA, and their polyplexes possessed enhanced colloidal stability in the presence of serum proteins. In vitro studies with mammalian CHO-1, 3T3, and Cos-7 cell lines demonstrated improved transfection efficiency of the pCMVbeta-gal plasmid/gPEI-Jeffamine polyplexes. The guanidinylation of the amino groups of PEI and the conjugation of PEI with the Jeffamine polyether enhanced the conjugates' interaction with genetic material and reduced the cytotoxicity of the polyplexes in experiments with the L929 cell line.

The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes : A comparative study

NARCIS (Netherlands)

Kneuer, Carsten; Ehrhardt, Carsten; Bakowsky, Heike; Kumar, M. N. V. Ravi; Oberle, Volker; Lehr, Claus M.; Hoekstra, Dick; Bakowsky, Udo

2006-01-01

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class

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

Adoptive T-cell therapy based on the infusion of patient’s own immune cells after ex vivo culturing is among the most potent forms of personalized treatment among recent clinical developments for the treatment of cancer. However, despite high rates of successful initial clinical responses, only...... 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...

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.

Induction of PLSCR1 in a STING/IRF3-dependent manner upon vector transfection in ovarian epithelial cells.

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Karthik M Kodigepalli

Full Text Available 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.

Stable convergence and stable limit theorems

CERN Document Server

Häusler, Erich

2015-01-01

The authors present a concise but complete exposition of the mathematical theory of stable convergence and give various applications in different areas of probability theory and mathematical statistics to illustrate the usefulness of this concept. Stable convergence holds in many limit theorems of probability theory and statistics – such as the classical central limit theorem – which are usually formulated in terms of convergence in distribution. Originated by Alfred Rényi, the notion of stable convergence is stronger than the classical weak convergence of probability measures. A variety of methods is described which can be used to establish this stronger stable convergence in many limit theorems which were originally formulated only in terms of weak convergence. Naturally, these stronger limit theorems have new and stronger consequences which should not be missed by neglecting the notion of stable convergence. The presentation will be accessible to researchers and advanced students at the master's level...

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

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Rezakhanlou Alireza

2010-03-01

Full Text Available Abstract Indoleamine 2,3 dioxygenase (IDO is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

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

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

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.

Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

DEFF Research Database (Denmark)

de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F

2014-01-01

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon po...

Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

Science.gov (United States)

Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

2016-01-01

Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture

International Nuclear Information System (INIS)

Martien, Ronny; Loretz, Brigitta; Sandbichler, Adolf Michael; Schnuerch, Andreas Bernkop

2008-01-01

The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 ± 86 and 113.6 ± 40 nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 ± 0.38 mV for unmodified chitosan nanoparticles and 4.3 ± 0.74 mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy

Influence of horse stable environment on human airways

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Pringle John

2009-05-01

Full Text Available Abstract Background Many people spend considerable amount of time each day in equine stable environments either as employees in the care and training of horses or in leisure activity. However, there are few studies available on how the stable environment affects human airways. This study examined in one horse stable qualitative differences in indoor air during winter and late summer conditions and assessed whether air quality was associated with clinically detectable respiratory signs or alterations to selected biomarkers of inflammation and lung function in stable personnel. Methods The horse stable environment and stable-workers (n = 13 in one stable were investigated three times; first in the winter, second in the interjacent late summer and the third time in the following winter stabling period. The stable measurements included levels of ammonia, hydrogen sulphide, total and respirable dust, airborne horse allergen, microorganisms, endotoxin and glucan. The stable-workers completed a questionnaire on respiratory symptoms, underwent nasal lavage with subsequent analysis of inflammation markers, and performed repeated measurements of pulmonary function. Results Measurements in the horse stable showed low organic dust levels and high horse allergen levels. Increased viable level of fungi in the air indicated a growing source in the stable. Air particle load as well as 1,3-β-glucan was higher at the two winter time-points, whereas endotoxin levels were higher at the summer time-point. Two stable-workers showed signs of bronchial obstruction with increased PEF-variability, increased inflammation biomarkers relating to reported allergy, cold or smoking and reported partly work-related symptoms. Furthermore, two other stable-workers reported work-related airway symptoms, of which one had doctor's diagnosed asthma which was well treated. Conclusion Biomarkers involved in the development of airway diseases have been studied in relation to

Influence of horse stable environment on human airways.

Science.gov (United States)

Elfman, Lena; Riihimäki, Miia; Pringle, John; Wålinder, Robert

2009-05-25

Many people spend considerable amount of time each day in equine stable environments either as employees in the care and training of horses or in leisure activity. However, there are few studies available on how the stable environment affects human airways. This study examined in one horse stable qualitative differences in indoor air during winter and late summer conditions and assessed whether air quality was associated with clinically detectable respiratory signs or alterations to selected biomarkers of inflammation and lung function in stable personnel. The horse stable environment and stable-workers (n = 13) in one stable were investigated three times; first in the winter, second in the interjacent late summer and the third time in the following winter stabling period. The stable measurements included levels of ammonia, hydrogen sulphide, total and respirable dust, airborne horse allergen, microorganisms, endotoxin and glucan. The stable-workers completed a questionnaire on respiratory symptoms, underwent nasal lavage with subsequent analysis of inflammation markers, and performed repeated measurements of pulmonary function. Measurements in the horse stable showed low organic dust levels and high horse allergen levels. Increased viable level of fungi in the air indicated a growing source in the stable. Air particle load as well as 1,3-beta-glucan was higher at the two winter time-points, whereas endotoxin levels were higher at the summer time-point. Two stable-workers showed signs of bronchial obstruction with increased PEF-variability, increased inflammation biomarkers relating to reported allergy, cold or smoking and reported partly work-related symptoms. Furthermore, two other stable-workers reported work-related airway symptoms, of which one had doctor's diagnosed asthma which was well treated. Biomarkers involved in the development of airway diseases have been studied in relation to environmental exposure levels in equine stables. Respirable dust and 1

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

International Nuclear Information System (INIS)

Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

1988-01-01

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

Science.gov (United States)

Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

2015-11-24

Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

Ballooning stable high beta tokamak equilibria

International Nuclear Information System (INIS)

Tuda, Takashi; Azumi, Masafumi; Kurita, Gen-ichi; Takizuka, Tomonori; Takeda, Tatsuoki

1981-04-01

The second stable regime of ballooning modes is numerically studied by using the two-dimensional tokamak transport code with the ballooning stability code. Using the simple FCT heating scheme, we find that the plasma can locally enter this second stable regime. And we obtained equilibria with fairly high beta (β -- 23%) stable against ballooning modes in a whole plasma region, by taking into account of finite thermal diffusion due to unstable ballooning modes. These results show that a tokamak fusion reactor can operate in a high beta state, which is economically favourable. (author)

Pharmaceuticals labelled with stable isotopes

International Nuclear Information System (INIS)

Krumbiegel, P.

1986-11-01

The relatively new field of pharmaceuticals labelled with stable isotopes is reviewed. Scientific, juridical, and ethical questions are discussed concerning the application of these pharmaceuticals in human medicine. 13 C, 15 N, and 2 H are the stable isotopes mainly utilized in metabolic function tests. Methodical contributions are given to the application of 2 H, 13 C, and 15 N pharmaceuticals showing new aspects and different states of development in the field under discussion. (author)

Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

NARCIS (Netherlands)

Wasungu, Luc; Scarzello, Marco; van Dam, Gooitzen; Molema, Grietje; Wagenaar, Anno; Engberts, Jan B. F. N.; Hoekstra, Dick

In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH

Myocardial perfusion SPECT in stable angina;Place de la scintigraphie myocardique dans l'angor stable

Energy Technology Data Exchange (ETDEWEB)

Jau, P.; Jacob, T. [HP Clairval, Service de medecine nucleaire, 13 - Marseille (France); Lecorff, G.; Bouvier, J.L.; Novella, P.; Bechet, V.; Pelet, V. [HP Clairval, Service de cardiologie, 13 - Marseille (France)

2010-03-15

We study the precise contribution of myocardial scintigraphy in the therapeutic management of stable coronary artery disease. Until recently, treatment was focused on revascularization, often by coronary angioplasty.Recent studies have challenged this practice by showing the absence of superiority of angioplasty compared to optimal medical therapy.The problem now is to define for each stable coronary artery disease, and individually, the best of both treatment options. In this spirit, the functional approach to coronary artery disease by myocardial perfusion scintigraphy is most interesting.The diagnostic performance, including sensitivity and negative predictive value, and the prognostic value of the technique are clearly established. Recent studies show that a therapeutic decision based on a functional approach to the patient is valid.We need to know this development in cardiology for best position in the multidisciplinary discussions, myocardial scintigraphy as a functional approach to stable coronary artery disease. (N.C.)

BRE enhances in vivo growth of tumor cells

International Nuclear Information System (INIS)

Chan, Ben Chung-Lap; Li Qing; Chow, Stephanie Ka-Yee; Ching, Arthur Kar-Keung; Liew, Choong Tsek; Lim, Pak-Leong; Lee, Kenneth Ka-Ho; Chan, John Yeuk-Hon; Chui, Y.-L.

2005-01-01

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

Science.gov (United States)

Kanani, S.; Pumir, A.; Krinsky, V.

2008-01-01

One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

Directory of Open Access Journals (Sweden)

Dae-Hee Sohn

Full Text Available An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs, recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs as stimulators of CD8(+ and CD4(+ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ enzyme-linked immunospot (ELISPOT assay by using isolated CD8(+ and CD4(+ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1-specific IFN-γ producing CD4(+ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+ T cells was significantly correlated with that of CD8(+ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001. To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+ T cell responses showed more significant changes than CD8(+ T cell responses. CD8(+ and CD4(+ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4 and Th17 (IL-17a cytokines were not detected. CD4(+ T cells secreted significantly higher cytokine levels than did CD8(+ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.

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

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Cecilia González

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

Adipose-derived stem cells transfected with pEGFP-OSX enhance bone formation during distraction osteogenesis*

OpenAIRE

Lai, Qing-guo; Sun, Shao-long; Zhou, Xiao-hong; Zhang, Chen-ping; Yuan, Kui-feng; Yang, Zhong-jun; Luo, Sheng-lei; Tang, Xiao-peng; Ci, Jiang-bo

2014-01-01

This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandib...

An Evaluation on Transfection Efficiency of pHRE-Egr1-EGFP in Hepatocellular Carcinoma Cells Bel-7402 Mediated by PEI-MZF-NPs

Directory of Open Access Journals (Sweden)

Mei Lin

2011-01-01

Full Text Available To improve transfection and expression efficiency of target gene, especially under cancer anoxic microenvironment, we have developed pHRE-Egr1-EGFP/PEI-MZF-NPs nanosystem, in which pHRE-Egr1-EGFP, eukaryotic gene expression plasmid, is constructed by combining radiation promoter Egr1 with anoxia induction components (HRE, forming anoxic radiation double sensitive HRE/Egr1 promoter to activate reporter gene EGFP expression. MZF-NPs (Mn0.5 Zn0.5 Fe2O4 magnetic nanoparticles, obtained by coprecipitation method, are coated with cation poly(ethylenimine (PEI. We transferred pHRE-Egr1-EGFP into hepatocellular carcinoma Bel-7402 cells, using PEI-MZF-NPs as the carrier and tested some relevant efficacy. The results show that PEI-MZF-NPs have good DNA-binding ability, protection ability, release ability, little toxicity, and high transfection efficiency, obviously superior to those of the liposome method and electricity perforation method. Moreover, the expression level of EGFP gene induced by anoxia and radiation was significantly higher than that of single radiation activation. It is therefore concluded that HRE/Egr1 can induce and improve target gene expression efficiency in cancer anoxic microenvironment, and that PEI-MZF-NPs can be used as a novel nonviral gene vector which offers a viable approach to the mediated radiation gene therapy of cancer.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

Science.gov (United States)

Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

2015-09-01

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can

The dissociation of tumor-induced weight loss from hypoglycemia in a transplantable pluripotent rat islet tumor results in the segregation of stable alpha- and beta-cell tumor phenotypes

DEFF Research Database (Denmark)

Madsen, O D; Karlsen, C; Nielsen, E

1993-01-01

in NEDH rats resulted in stable hypoglycemic insulinoma tumor lines, such as MSL-G2-IN. Occasionally, hypoglycemia as well as severe weight loss were observed in the early tumor passages of MSL-G and the subclone, NHI-5B, which carry the transfected neomycin and human insulin genes as unique clonal...... markers. By selective transplantation, it was possible to segregate stable anorectic normoglycemic tumor lines, MSL-G-AN and NHI-5B-AN, from both clones. These tumors cause an abrupt onset of anorexia when they reach a size of 400-500 mg (loss parallels...... a common clonal origin of pluripotent MSL cells, thus supporting the existence of a cell lineage relationship between islet alpha- and beta-cell during ontogeny; and 2) that our glucagonomas release an anorexigenic substance(s) of unknown nature that causes a severe weight loss comparable to that reported...

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

Science.gov (United States)

Rinehart, C A; Mayben, J P; Butler, T D; Haskill, J S; Kaufman, D G

1992-01-01

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

International Nuclear Information System (INIS)

Stark, M.; Naiman, T.; Canaani, D.

1989-01-01

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [ 3 H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line

Plasmid DNA transfection using magnetite cationic liposomes for construction of multilayered gene-engineered cell sheet.

Science.gov (United States)

Ino, Kosuke; Kawasumi, Tamayo; Ito, Akira; Honda, Hiroyuki

2008-05-01

Modification of cellular functions by overexpression of genes is being increasingly practiced for tissue engineering. In the present study, we investigated whether transfection efficiency could be enhanced by magnetofection that involves the use of plasmid DNA (pDNA)/magnetite cationic liposomes (MCLs) complexes (pDNA/MCL) and magnetic force. The transfection efficiencies of the magnetofection technique by pDNA/MCL in fibroblasts and keratinocytes using reporter genes were 36- and 10-fold higher, respectively, than those of a lipofection technique by cationic liposomes. Moreover, in vitro construction of three-dimensional (3D) tissues is an important challenge. We recently proposed a novel technique termed "magnetic force-based tissue engineering" (Mag-TE) to produce 3D tissues. Since the fibroblasts after magnetofection incorporated both magnetite nanoparticles and pDNA, we investigated whether multilayered heterotypic cell sheets expressing transgene could be fabricated by Mag-TE. First, the fibroblasts were seeded onto an ultra-low attachment culture plate. When a magnet was placed under the plate, the cells accumulated at the bottom of the culture plate. After 24 h of culture, the transgene-expressing cells formed a multilayered cell sheet-like structure. These results indicated that MCLs are a potent biomanipulation tool for both gene transfer and 3D tissue construction, suggesting that these techniques are useful for tissue engineering. Copyright 2007 Wiley Periodicals, Inc.

Composites of malonic acid diamides and phospholipids--Impact of lipoplex stability on transfection efficiency.

Science.gov (United States)

Janich, Christopher; Wölk, Christian; Erdmann, Frank; Groth, Thomas; Brezesinski, Gerald; Dobner, Bodo; Langner, Andreas

2015-12-28

The use of cationic lipids as gene delivery systems is a basic method in gene therapy. Through ongoing research, lipofection is currently the leader of non-viral vectors in clinical trials. However, in order to unleash the full potential of lipofection further intensive investigations are indispensable. In this study, various lipoplex formulations were compared regarding their ability to bind DNA. To obtain information about a possible premature release of DNA at the cell surface, heparin and chondroitin dependent lipoplex destabilization experiments were carried out. Complementary investigations in cell culture were performed to quantify DNA outside the cell. Additionally, DNase I stability was investigated. In this regard a multitude of methods, namely confocal laser scanning microscopy (CLSM), polymerase chain reaction (PCR), cell culture experiments, ethidium bromide assay, gel electrophoresis, Langmuir-isotherm experiments, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), zeta-(ζ)-potential measurements, and dynamic light scattering (DLS), were applied. Although the complexation of DNA is a fundamental step, we show that the DNA release by biological agents (proteoglycans) and an unsuccessful cell attachment are major transfection limiting parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

Science.gov (United States)

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

2017-11-01

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Non-linear Yang-Mills instantons from strings are π-stable D-branes

International Nuclear Information System (INIS)

Enger, H.; Luetken, C.A.

2004-01-01

We show that B-type Π-stable D-branes do not in general reduce to the (Gieseker-) stable holomorphic vector bundles used in mathematics to construct moduli spaces. We show that solutions of the almost Hermitian Yang-Mills equations for the non-linear deformations of Yang-Mills instantons that appear in the low-energy geometric limit of strings exist iff they are π-stable, a geometric large volume version of Π-stability. This shows that π-stability is the correct physical stability concept. We speculate that this string-canonical choice of stable objects, which is encoded in and derived from the central charge of the string-algebra, should find applications to algebraic geometry where there is no canonical choice of stable geometrical objects

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

NARCIS (Netherlands)

Yang, X.; Walboomers, X.F.; Dolder, J. van den; Yang, F.; Bian, Z.; Fan, M.; Jansen, J.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

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes-A model

Energy Technology Data Exchange (ETDEWEB)

Kanani, S. [Institut Genomique Fonctionelle, 141 Rue de la Cardonille, 34396 Montpellier (France); Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France); Pumir, A. [Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France); Laboratoire J.A. Dieudonne, CNRS and Universite de Nice, Parc Valrose, 06108 Nice (France)], E-mail: alain.pumir@unice.fr; Krinsky, V. [Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France)

2008-01-07

One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler-Reuter model, as well as with the elaborate dynamic Luo-Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin-Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I{sub K1} channels is low enough. At too high an expression level of I{sub K1} channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I{sub K1} channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I{sub K1} channels observed in ventricular myocytes, both in the Beeler-Reuter and in the dynamic Luo-Rudy models are too high to allow to observe oscillations. With expression levels below {approx}1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I{sub K1} has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

Stable isotopes

International Nuclear Information System (INIS)

Evans, D.K.

1986-01-01

Seventy-five percent of the world's stable isotope supply comes from one producer, Oak Ridge Nuclear Laboratory (ORNL) in the US. Canadian concern is that foreign needs will be met only after domestic needs, thus creating a shortage of stable isotopes in Canada. This article describes the present situation in Canada (availability and cost) of stable isotopes, the isotope enrichment techniques, and related research programs at Chalk River Nuclear Laboratories (CRNL)

Moving stable solitons in Galileon theory

International Nuclear Information System (INIS)

Masoumi, Ali; Xiao Xiao

2012-01-01

Despite the no-go theorem Endlich et al. (2011) which rules out static stable solitons in Galileon theory, we propose a family of solitons that evade the theorem by traveling at the speed of light. These domain-wall-like solitons are stable under small fluctuations-analysis of perturbation shows neither ghost-like nor tachyon-like instabilities, and perturbative collision of these solitons suggests that they pass through each other asymptotically, which maybe an indication of the integrability of the theory itself.

Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of patients with malignant gliomas

Directory of Open Access Journals (Sweden)

Torres-Trejo Alejandro

2007-12-01

Full Text Available Abstract Background The prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL-4 gene transfected fibroblasts. Methods In University of Pittsburgh Cancer Institute (UPCI protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM or anaplastic astrocytoma (AA received gross total resection (GTR of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging. Results and Discussion In UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN-γ Enzyme-Linked Immuno-SPOT (ELISPOT assay in another human leukocyte antigen (HLA-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA epitope EphA2883–891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants

Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines.

Science.gov (United States)

Roukaerts, Inge D M; Grant, Chris K; Theuns, Sebastiaan; Christiaens, Isaura; Acar, Delphine D; Van Bockstael, Sebastiaan; Desmarets, Lowiese M B; Nauwynck, Hans J

2017-01-02

Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission. Copyright © 2016. Published by Elsevier B.V.

Stable Isotope Data

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Tissue samples (skin, bone, blood, muscle) are analyzed for stable carbon, stable nitrogen, and stable sulfur analysis. Many samples are used in their entirety for...

stableGP

Data.gov (United States)

National Aeronautics and Space Administration — The code in the stableGP package implements Gaussian process calculations using efficient and numerically stable algorithms. Description of the algorithms is in the...

Tumor transfection after systemic injection of DNA lipid nanocapsules.

Science.gov (United States)

Morille, Marie; Passirani, Catherine; Dufort, Sandrine; Bastiat, Guillaume; Pitard, Bruno; Coll, Jean-Luc; Benoit, Jean-Pierre

2011-03-01

With the goal of generating an efficient vector for systemic gene delivery, a new kind of nanocarrier consisting of lipid nanocapsules encapsulating DOTAP/DOPE lipoplexes (DNA LNCs) was pegylated by the post-insertion of amphiphilic and flexible polymers. The aim of this surface modification was to create a long-circulating vector, able to circulate in the blood stream and efficient in transfecting tumoral cells after passive targeting by enhanced permeability and retention effect (EPR effect). PEG conformation, electrostatic features, and hydrophylicity are known to be important factors able to influence the pharmacokinetic behaviour of vectors. In this context, the surface structure characteristics of the newly pegylated DNA LNCs were studied by measuring electrophoretic mobility as a function of ionic strength in order to establish a correlation between surface properties and in vivo performance of the vector. Finally, thanks to this PEGylation, gene expression was measured up to 84-fold higher in tumor compared to other tested organs after intravenous injection. The present results indicate that PEGylated DNA LNCs are promising carriers for an efficient cancer gene therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

Solutions for the stable roommates problem with payments

NARCIS (Netherlands)

Biró, Péter; Bomhoff, M.J.; Golovach, Petr A.; Kern, Walter

2014-01-01

The stable roommates problem with payments has as input a graph G = (V , E ) with an edge weighting w : E → R≥0 and the problem is to find a stable solution. By pinpointing a relationship to the accessibility of the coalition structure core of matching games, we give a constructive proof for showing

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

Science.gov (United States)

Guo, Xiaodong; Zheng, Qixin; Kulbatski, Iris; Yuan, Quan; Yang, Shuhua; Shao, Zengwu; Wang, Hong; Xiao, Baojun; Pan, Zhengqi; Tang, Shuo

2006-09-01

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 b

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

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous β-TCP ceramic scaffolds

International Nuclear Information System (INIS)

Guo Xiaodong; Zheng Qixin; Kulbatski, Iris; Yuan Quan; Yang Shuhua; Shao Zengwu; Wang Hong; Xiao Baojun; Pan Zhengqi; Tang Shuo

2006-01-01

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 β tricalcium phosphate (β-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 b

Stability properties of nonlinear dynamical systems and evolutionary stable states

Energy Technology Data Exchange (ETDEWEB)

Gleria, Iram, E-mail: iram@fis.ufal.br [Instituto de Física, Universidade Federal de Alagoas, 57072-970 Maceió-AL (Brazil); Brenig, Leon [Faculté des Sciences, Université Libre de Bruxelles, 1050 Brussels (Belgium); Rocha Filho, Tarcísio M.; Figueiredo, Annibal [Instituto de Física and International Center for Condensed Matter Physics, Universidade de Brasília, 70919-970 Brasília-DF (Brazil)

2017-03-18

Highlights: • We address the problem of equilibrium stability in a general class of non-linear systems. • We link Evolutionary Stable States (ESS) to stable fixed points of square quasi-polynomial (QP) systems. • We show that an interior ES point may be related to stable interior fixed points of QP systems. - Abstract: In this paper we address the problem of stability in a general class of non-linear systems. We establish a link between the concepts of asymptotic stable interior fixed points of square Quasi-Polynomial systems and evolutionary stable states, a property of some payoff matrices arising from evolutionary games.

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

[Experimental study on human periodontal ligament cells transfected with human amelogenin gene].

Science.gov (United States)

Yu, Guang; Shu, Rong; Sun, Ying; Cheng, Lan; Song, Zhong-Chen; Zhang, Xiu-Li

2008-02-01

To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction. The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR). The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR. The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).

COBRA1 inhibits AP-1 transcriptional activity in transfected cells

International Nuclear Information System (INIS)

Zhong Hongjun; Zhu Jianhua; Zhang Hao; Ding Lihua; Sun Yan; Huang Cuifen; Ye Qinong

2004-01-01

Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis

Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

Science.gov (United States)

Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-01-01

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

Establishment and evaluation of a stable cattle type II alveolar epithelial cell line.

Directory of Open Access Journals (Sweden)

Feng Su

Full Text Available Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

Science.gov (United States)

Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

2014-07-01

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

Science.gov (United States)

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

International Nuclear Information System (INIS)

Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang; Ito, Etsuro; Bertino, J.R.

1988-01-01

A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [ 14 C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14 C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14 C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice

Pathological study on the testis of mice irradiated by γ-rays after transfecting pprI gene by in vivo electroporation

International Nuclear Information System (INIS)

Lian Lixia; Chen Tingting; Zhang Yongqin; Wang Xiuzhen; Yang Zhanshan

2011-01-01

reduction of spermatogonia was generally marked, and the number of spermatogenic cells reduced. By the 28th day some spermatogonia were actively regenerating in some of the tubules. By the 35th day the regeneration of spermatogonia increased and began to stratify. The expression of the pprI gene transfected by in vivo electroporation in mice irradiated by γ-ray shows an significant anti-radiation effect, and can promote the reparative process of testis injury from the mice. (authors)

On some topological properties of stable measures

DEFF Research Database (Denmark)

Nielsen, Carsten Krabbe

1996-01-01

Summary The paper shows that the set of stable probability measures and the set of Rational Beliefs relative to a given stationary measure are closed in the strong topology, but not closed in the topology of weak convergence. However, subsets of the set of stable probability measures which...... are characterized by uniformity of convergence of the empirical distribution are closed in the topology of weak convergence. It is demonstrated that such subsets exist. In particular, there is an increasing sequence of sets of SIDS measures who's union is the set of all SIDS measures generated by a particular...... system and such that each subset consists of stable measures. The uniformity requirement has a natural interpretation in terms of plausibility of Rational Beliefs...

Accumulation of a soluble form of human nectin-2 is required for exerting the resistance against herpes simplex virus type 2 infection in transfected cells.

Science.gov (United States)

Fujimoto, Y; Ozaki, K; Iwamori, N; Takakuwa, H; Ono, E

2016-03-01

Cell entry of herpes simplex virus type 2 (HSV-2) requires the interaction of viral glycoprotein D (gD) with the receptor nectin-1 and herpesvirus entry mediator (HVEM). In addition, it is known that nectin-2 is also functional as a receptor for HSV-2, although the binding to the gD is weak. To examine an antiviral potential of a soluble form of human nectin-2 (hNectin-2Ig), transfected Vero cells expressing the entire ectodomain of nectin-2 fused to the Fc portion of human IgG were established. Specific binding of hNectin-2Ig to HSV-2 gD was confirmed by ELISA. Competitive ELISA demonstrated that accumulation of hNectin-2Ig in transfected cells increased significantly in a cell culture time dependent manner. Viral growth of several HSV-2 strains was significantly inhibited in the transfected cells that were cultured for 72 hr compared with control Vero cells, but not in cells that were cultured for 24 hr. These results indicate that accumulation of a soluble form of nectin-2 is required for exerting the resistance against HSV-2 infection.

Study of multidrug resistance and radioresistance

International Nuclear Information System (INIS)

Kang, Yoon Koo; Yoo, Young Do

1999-04-01

We investigated the mechanism of 5-FU, adriamycin, radiation resistance in Korean gastric cancer cells. First we investigated the relation between Rb and multidrug resistance. Rb stable transfectants exhibited 5- to 10- fold more resistance to adriamycin than the control cells. These Rb transfectants showed increased MDR1 expression. We also investigated up-regulation in radiation-resistant tumor tissues. HSP27, MRP-8, GST, and NKEF-B were up-regulated in radiation resistant tumor. Expression of NKEF-B was also increased by radiation exposure in Head and Neck cells. These results demonstrated that NKEF-B is a stress response protein and it may have an important role in radiation resistance

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

Directory of Open Access Journals (Sweden)

Alexander Raup

2016-06-01

Full Text Available Genetic modification (“transfection” of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino ethyl methacrylate (DMAEMA building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293 and non-adherent (Jurkat, primary human T lymphocytes mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities had been grown from a center silsesquioxane or silica-coated γ-Fe2O3-core and one micellar structure self-assembled from poly(1,2-butadiene-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate, provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution, but also the concentrations adjusted during polyplex formation, affected the results.

Stable isotopes

International Nuclear Information System (INIS)

Brazier, J.L.; Guinamant, J.L.

1995-01-01

According to the progress which has been realised in the technology of separating and measuring isotopes, the stable isotopes are used as preferable 'labelling elements' for big number of applications. The isotopic composition of natural products shows significant variations as a result of different reasons like the climate, the seasons, or their geographic origins. So, it was proved that the same product has a different isotopic composition of alimentary and agriculture products. It is also important in detecting the pharmacological and medical chemicals. This review article deals with the technology, like chromatography and spectrophotometry, adapted to this aim, and some important applications. 17 refs. 6 figs

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection.

Science.gov (United States)

Van Pham, Phuc; Thi-My Nguyen, Phuoc; Thai-Quynh Nguyen, Anh; Minh Pham, Vuong; Nguyen-Tu Bui, Anh; Thi-Tung Dang, Loan; Gia Nguyen, Khue; Kim Phan, Ngoc

2014-06-01

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression

International Nuclear Information System (INIS)

Patrick, Brad; Li Jie; Jeyabal, Prince V.S.; Reddy, Prasada M.R.V.; Yang Yusong; Sharma, Rajendra; Sinha, Mala; Luxon, Bruce; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

2005-01-01

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960-1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin γ1, connexin 43, Fas, integrin α6, TGFα, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCβII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFβ was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions

Establishment, characterization, virus susceptibility and transfection of cell lines from cobia, Rachycentron canadum (L.), brain and fin.

Science.gov (United States)

Cheng, T-C; Lai, Y-S; Lin, I-Y; Wu, C-P; Chang, S-L; Chen, T-I; Su, M-S

2010-02-01

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate-buffered saline containing 0.25% trypsin at 25 degrees C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 degrees C in Leibovitz-15 medium containing 10% foetal bovine serum. The cells have been sub-cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.

A parallel approach to the stable marriage problem

DEFF Research Database (Denmark)

Larsen, Jesper

1997-01-01

This paper describes two parallel algorithms for the stable marriage problem implemented on a MIMD parallel computer. The algorithms are tested against sequential algorithms on randomly generated and worst-case instances. The results clearly show that the combination fo a very simple problem...... and a commercial MIMD system results in parallel algorithms which are not competitive with sequential algorithms wrt. practical performance. 1 Introduction In 1962 the Stable Marriage Problem was....

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Institute of Scientific and Technical Information of China (English)

Jielu Tan; Xiangrong Zheng; Shanshan Zhang; Yujia Yang; Xia Wang; Xiaohe Yu; Le Zhong

2014-01-01

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge-nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into ifve groups: (1) sham operation (control), (2) cerebral palsy model alone or with (3) phosphate-buffered saline, (4) vascular en-dothelial growth factor 165 + neural stem cells, or (5) neural stem cells alone. hTe cerebral palsy model was established by ligating the letf common carotid artery followed by exposure to hypox-ia. Phosphate-buffered saline, vascular endothelial growth factor + neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. Atfer transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas-cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for ifnding water and the ifnding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. hTese ifndings indicate that the transplantation of vascu-lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deifcits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Hua, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Fang, Luan [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Ying, Ju [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Hongyu, Shen [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lifen, Gao [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Xiaoyan, Wang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Suxia, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lining, Zhang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Wensheng, Sun [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Chunhong, Ma [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Key Laboratory for Experimental Teratology, Ministry of Education (China)]. E-mail: machunhong@sdu.edu.cn

2007-04-06

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

Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

Science.gov (United States)

Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

2014-09-01

Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients.

On Nash-Equilibria of Approximation-Stable Games

Science.gov (United States)

Awasthi, Pranjal; Balcan, Maria-Florina; Blum, Avrim; Sheffet, Or; Vempala, Santosh

One reason for wanting to compute an (approximate) Nash equilibrium of a game is to predict how players will play. However, if the game has multiple equilibria that are far apart, or ɛ-equilibria that are far in variation distance from the true Nash equilibrium strategies, then this prediction may not be possible even in principle. Motivated by this consideration, in this paper we define the notion of games that are approximation stable, meaning that all ɛ-approximate equilibria are contained inside a small ball of radius Δ around a true equilibrium, and investigate a number of their properties. Many natural small games such as matching pennies and rock-paper-scissors are indeed approximation stable. We show furthermore there exist 2-player n-by-n approximation-stable games in which the Nash equilibrium and all approximate equilibria have support Ω(log n). On the other hand, we show all (ɛ,Δ) approximation-stable games must have an ɛ-equilibrium of support O(Δ^{2-o(1)}/ɛ2{log n}), yielding an immediate n^{O(Δ^{2-o(1)}/ɛ^2log n)}-time algorithm, improving over the bound of [11] for games satisfying this condition. We in addition give a polynomial-time algorithm for the case that Δ and ɛ are sufficiently close together. We also consider an inverse property, namely that all non-approximate equilibria are far from some true equilibrium, and give an efficient algorithm for games satisfying that condition.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

Science.gov (United States)

Domarkas, Juozas; Dudouit, Fabienne; Williams, Christopher; Qiyu, Qiu; Banerjee, Ranjita; Brahimi, Fouad; Jean-Claude, Bertrand Jacques

2006-06-15

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

Agonist/antagonist interactions with cloned human 5-HT(1A) receptors: Variations in intrinsic activity studied in transfected HeLa cells

NARCIS (Netherlands)

Boddeke, H.W.G.M.; Fargin, A.; Raymond, J.R.; Schoeffter, P.; Hoyer, D.

1992-01-01

The characteristics of 5-HT(1A)-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT(1A) receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond

Direct transition from a stable equilibrium to quasiperiodicity in non-smooth systems

DEFF Research Database (Denmark)

Zhusubaliyev, Z.T.; Mosekilde, Erik

2008-01-01

The purpose of this Letter is to show how a border-collision bifurcation in a piecewise-smooth dynamical system can produce a direct transition from a stable equilibrium point to a two-dimensional invariant torus. Considering a system of nonautonomous differential equations describing the behavior...... of a power electronic DC/DC converter, we first determine the chart of dynamical modes and show that there is a region of parameter space in which the system has a single stable equilibrium point. Under variation of the parameters, this equilibrium may collide with a discontinuity boundary between two smooth...... regions in phase space. When this happens, one can observe a number of different bifurcation scenarios. One scenario is the continuous transformation of the stable equilibrium into a stable period-1 cycle. Another is the transformation of the stable equilibrium into an unstable period-1 cycle with complex...

Heat-stable proteins and abscisic acid action in barley aleurone cells

International Nuclear Information System (INIS)

Jacobsen, J.V.; Shaw, D.C.

1989-01-01

[ 35 S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degree C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heat-stable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered

Transfection of wild type ADVP53 gene into human brain tumor cell lines has a radiosensitizing effect independent of apoptosis

International Nuclear Information System (INIS)

Geng, L.; Walter, S; Vaughan, A.T.M.

1997-01-01

Purpose: Despite attempts with a variety of therapeutic approaches there has been little impact on the survival of patients with Glioblastoma multiforme, with median survivals reported of approximately 12 months. In this study a replication restricted adenovirus vector is used to transfer the wild type p53 gene into two cell lines derived from a human astrocytoma U87MG or glioblastoma T98G, to determine its ability to act as a radiosensitizer in conjunction with conventional radiotherapy. Methods: An adenovirus vector containing the human wild type p53 (Advp53) gene was used in addition to a control vector containing the β-galactosidase (Advγgal) reporter gene. To achieve cellular incorporation both vectors were incubated with cells for 30 minutes - washed and returned to culture. The successful incorporation of each vector was determined by either a p53 assay using either a western blotting or flow cytometry techniques, or specific staining for β-galactosidase activity. The presence of each vector was assayed until the constructs were eliminated from the cell. To determine the effects of these vectors on cell survival sufficient vector was added to produce a measurable reduction in clonogenic survival and this value was used in subsequent irradiation experiments. To determine the ability of wild type p53 to induce apoptosis the cells were examined from 1 to 5 days after irradiation by H and E staining for the characteristic morphology indicating an apoptotic process. Results: Both the Advp53 and Advβgal vectors were successfully incorporated into each cell line. Expression of each gene was reduced to approximately half by 5 days and virtually eliminated by 15 days after transfection in both lines. At the doses used the wild type Advp53 adenovirus was toxic to both cell lines giving surviving fractions between 39-74%. When this toxicity was taken into account the presence of the Advp53 gene had a radiosensitizing effect in each cell line. To determine the

Malignant transformation of diploid human fibroblasts by transfection of oncogenes: Progress report, July 1986--June 1989

International Nuclear Information System (INIS)

McCormick, J.J.; Maher, V.M.

1989-01-01

Although there is good evidence that carcinogen exposure is a major cause of human cancer, it has proven impossible to transform normal human fibroblasts or epithelial cells in culture into malignant cells by treating them with carcinogens. This failure may reflect an inability to identify and isolate cells containing one or more premalignant changes so that these can be expanded and exposed to carcinogens a second time to induce additional required changes. A second serious roadblock to the sequential introduction of changes and expansion of clonally-derived cells containing such premalignant changes in the finite life span of human cells in culture. Using transfection of specific human oncogenes in a series of specially-selected vectors, we have overcome these obstacles and have recently succeeded in generating an infinite life span diploid human cell strain MSU-1.0, which appears to be normal in all other characteristics. From that cell a second cell strain, MSU-1.1, was generated which we have been able to transform into a malignant state not only by transfecting the cells with oncogenes but also by treating them with chemical carcinogens. We now have evidence that there is not just a single linear process which results in malignant transformation. Rather, cells appear to progress to malignancy on a series of parallel, sometimes overlapping tracks. We now propose to carry out detailed studies of the specific mechanisms of malignant cell transformation using the cell strains available in this laboratory to achieve the goal of building relevant quantitative models of carcinogenesis. 29 refs

DFT computations of the lattice constant, stable atomic structure and ...

African Journals Online (AJOL)

This paper presents the most stable atomic structure and lattice constant of Fullerenes (C60). FHI-aims DFT code was used to predict the stable structure and the computational lattice constant of C60. These were compared with known experimental structures and lattice constants of C60. The results obtained showed that ...

Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor {beta}{sub 1} gene

Energy Technology Data Exchange (ETDEWEB)

Guo Xiaodong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng Qixin [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yang Shuhua [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Shao Zengwu [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yuan Quan [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Pan Zhengqi [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Tang Shuo [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Liu Kai [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Quan Daping [Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275 (China)

2006-12-15

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-{beta}{sub 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-{beta}{sub 1} that may circumvent difficulties associated with growth factor delivery. The objective of this study was to investigate whether TGF-{beta}{sub 1} gene modified mesenchymal stem cells (MSCs) could enhance the repair of full-thickness articular cartilage defects in allogeneic rabbits. The pcDNA{sub 3}-TGF-{beta}{sub 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{sub 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

PHARMACOLOGICAL IN VITRO MODELS IN PRE-CLINICAL DRUG TESTING - EXAMPLE OF hSERT TRANSFECTED HUMAN EMBRYONIC KIDNEY CELLS

Directory of Open Access Journals (Sweden)

Mihajlo Jakovljević

2012-06-01

Full Text Available Preclinical drug testing should be considered an important stage during examinations of its efficiency and safety in any likely indication observed. Purpose of the process is acquisition of substantial amount of particular drug-related data before approaching clinical trials in humans. Historical preclinical testing relied on available testing in microbe cultures and animal models. During recent decades laboratory techniques of human cell lines cultivation have been developed and improved. These provide unique possibility of drug acting mechanism testing in a simplified environment lacking basic homeostatic mechanisms. Some examples of these are measuring drug impact to biochemical transport, signaling or anabolic processes. Humane cell lines of embrional kidney 293 are an example of easy-to-grow and disseminate and quite endurable cell line. This methodological article notices some of the details of HEK293 cells cultivation and breading. We took transfection as an example of in vitro model creation for drug testing. Transfection refers to gene introduction into HEK293 cellular genome in order to achieve membrane expression of coded protein. In our case it would be human serotonin transporter. Article contains description of one particular methodological approach in measuring human serotonin transporter expression. The role and importance of serotonin pump in affective disorders genesis was already widely recognized. Aim of the paper was to emphasize feasibility of cell cultivation and its advantages in comparison with alternative traditional methods.

One-dimensional stable distributions

CERN Document Server

Zolotarev, V M

1986-01-01

This is the first book specifically devoted to a systematic exposition of the essential facts known about the properties of stable distributions. In addition to its main focus on the analytic properties of stable laws, the book also includes examples of the occurrence of stable distributions in applied problems and a chapter on the problem of statistical estimation of the parameters determining stable laws. A valuable feature of the book is the author's use of several formally different ways of expressing characteristic functions corresponding to these laws.

Bi-stable optical actuator

Science.gov (United States)

Holdener, Fred R.; Boyd, Robert D.

2000-01-01

The present invention is a bi-stable optical actuator device that is depowered in both stable positions. A bearing is used to transfer motion and smoothly transition from one state to another. The optical actuator device may be maintained in a stable position either by gravity or a restraining device.

Evolutionary Stable Strategy

Indian Academy of Sciences (India)

Home; Journals; Resonance – Journal of Science Education; Volume 21; Issue 9. Evolutionary Stable Strategy: Application of Nash Equilibrium in Biology. General Article Volume 21 Issue 9 September 2016 pp 803- ... Keywords. Evolutionary game theory, evolutionary stable state, conflict, cooperation, biological games.

Axisymmetric MHD stable sloshing ion distributions

International Nuclear Information System (INIS)

Berk, H.L.; Dominguez, N.; Roslyakov, G.V.

1986-07-01

The MHD stability of a sloshing ion distribution is investigated in a symmetric mirror cell. Fokker-Planck calculations show that stable configurations are possible for ion injection energies that are at least 150 times greater than the electron temperture. Special axial magnetic field profiles are suggested to optimize the favorable MHD properties

On the Restriction of the Location of Stable Points for Generalized Lotka-Volterra

OpenAIRE

Livesay, Michael Richard

2017-01-01

We develop tools to determine which fixed points in a generalized Lotka-Volterra system are stable, under certain non-degeneracy conditions. We characterize which faces of the boundary of the domain of the Lotka-Volterra system could contain a stable fixed point. Under various relaxed conditions, we show that whenever a face of the boundary contains a stable point there are no other stable points in any strictly larger face of the boundary.

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.

The Proteolytically Stable Peptidomimetic Pam-(Lys-ßNSpe)6-NH2 Selectively Inhibits Human Neutrophil Activation via Formyl Peptide Receptor 2

DEFF Research Database (Denmark)

Skovbakke, Sarah Line; Heegaard, Peter M. H.; Larsen, Camilla J.

2015-01-01

of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release...... of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogues of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging...... flow cytometry in primary neutrophils and FPR-transfected cell lines we found that a fluorescently labelled analogue of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating...

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.

Stable isotope probing of acetate fed anaerobic batch incubations shows a partial resistance of acetoclastic methanogenesis catalyzed by Methanosarcina to sudden increase of ammonia level.

Science.gov (United States)

Hao, Liping; Lü, Fan; Mazéas, Laurent; Desmond-Le Quéméner, Elie; Madigou, Céline; Guenne, Angéline; Shao, Liming; Bouchez, Théodore; He, Pinjing

2015-02-01

Ammonia inhibition represents a major operational issue for anaerobic digestion. In order to refine our understanding of the terminal catabolic steps in thermophilic anaerobic digestion under ammonia stress, we studied batch thermophilic acetate fed experiments at low (0.26 g L(-1)) and high (7.00 g L(-1)) Total Ammonia Nitrogen concentrations (TAN). Although methane production started immediately for all incubations and resulted in methane yields close to stoichiometric expectations, a 62-72% decrease of methanogenic rate was observed throughout the incubation at 7.00 g L(-1) of TAN compared to 0.26 g L(-1). Stable Isotope Probing analysis of active microbial communities in (13)C-acetate fed experiments coupled to automated ribosomal intergenic spacer analysis and 16S rDNA pyrotag sequencing confirmed that microbial communities were similar for both TAN conditions. At both TAN levels, the (13)C-labeled bacterial community was mainly affiliated to Clostridia-relatives, with OPB54 bacteria being the most abundant sequence in the heavy DNA 16S rDNA pyrotag library. Sequences closely related to Methanosarcina thermophila were also abundantly retrieved in the heavy DNA fractions, showing that this methanogen was still actively assimilating labeled carbon from acetate at free ammonia nitrogen concentrations up to 916 mg L(-1). Stable isotopic signature analysis of biogas, measured in unlabeled acetate fed experiments that were conducted in parallel, confirmed that acetoclastic methanogenic pathway was dominant at both ammonia concentrations. Our work demonstrates that, besides the syntrophic acetate oxidation pathway, acetoclastic methanogenesis catalyzed by Methanosarcina can also play a major role in methane production at high ammonia levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro

NARCIS (Netherlands)

Fielden, Matthew; Perrin, C.; Kremer, Andreas; Bergsma, M.; Stuart, M.C.A.; Camilleri, P.; Engberts, J.B.F.N.

2001-01-01

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12-18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1).

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection

Directory of Open Access Journals (Sweden)

Yan Wang

2017-02-01

Full Text Available CD4+CD25+Foxp3+ T regulatory (Treg cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+ cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

Role of integrin-linked kinase for functional capacity of endothelial progenitor cells in patients with stable coronary artery disease

International Nuclear Information System (INIS)

Werner, Christian; Boehm, Michael; Friedrich, Erik B.

2008-01-01

Number and function of endothelial progenitor cells (EPCs) are down-regulated in patients with coronary artery disease (CAD). Integrin-linked kinase (ILK) is a signal and adaptor protein that regulates survival of mature endothelial cells and vascular development. Here we show that EPC dysfunction in patients with CAD is paralleled by down-regulation of ILK while restoration of ILK expression rescues the migratory defect of CAD-EPCs. Human EPCs transduced with dominant-negative ILK (DN-ILK) display significantly reduced expression of CD34 + /VEGFR-2 + , DiI-Ac-LDL uptake, and Ulex europaeus lectin binding. Mechanistically, DN-ILK-transfected EPCs are characterized by decreased proliferation, while proliferation is increased in wild-type ILK-transfected EPCs. These effects are paralleled by changes in cyclin D1 expression, colony forming units, and cytoskeletal rearrangement. Functionally, ILK is necessary and sufficient for SDF-1-triggered migration and adhesion in EPCs. These data extend current knowledge about the role of ILK in EPC biology and implicate ILK as a therapeutic target in CAD.

Synthesis and evaluation of tetramethylguanidinium-polyethylenimine polymers as efficient gene delivery vectors.

Science.gov (United States)

Mahato, Manohar; Yadav, Santosh; Kumar, Pradeep; Sharma, Ashwani Kumar

2014-01-01

Previously, we demonstrated that 6-(N,N,N',N'-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers exhibited significantly enhanced transfection efficiency and cell viability. Here, in the present study, we have synthesized a series of N,N,N',N'-tetramethylguanidinium-polyethylenimine (TP1-TP5) polymers via a single-step reaction involving peripheral primary amines of bPEI and varying amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). These polymers were found to interact efficiently with negatively charged pDNA and formed stable complexes in the size range of ~240-450 nm. Acid-base titration profiles revealed improved buffering capacity of TP polymers as compared to bPEI. Transfection and cytotoxicity assays performed with TP/pDNA complexes on HEK293, CHO, and HeLa cells showed significantly higher transfection efficiency and cell viability with one of the complexes, TP2/pDNA complex, exhibited the highest transfection efficiency (~1.4-2.3-fold) outcompeting native bPEI and the commercially available transfection reagent, Lipofectamine 2000. Compared to previously reported THP polymers, the transfection efficiency of TP/pDNA complexes was found to be lower, as examined by flow cytometry. These results highlight the importance of the hydrophobic C-6 linker in THP polymers in forming compact nanostructures with pDNA, which might lead to efficient uptake and internalization of the complexes; however, the projected TP polymers offer an advantage of their rapid and economical one-step synthesis.

The effect of ionizing radiation and bleomycin on transfecting ability of Bacillus subtilis phage DNA

International Nuclear Information System (INIS)

Schafers, F.; Kohnlein, W.

1979-01-01

Infectious DNA of Bacillus subtilis phage 029 and SPP1 has been subjected to ionizing radiation and/or bleomycin treatment. The extent of degradation of the treated DNA was determined on sucrose gradients and biological activity was analyzed using the transfection principle. It was found that loss of biological activity following irradiation or bleomycin treatment of the DNA cannot be accounted for by the production of single- or double-strand breaks. Furthermore, it was observed that pre-irradiation exhibits a synergistic effect on loss of biological activity and production of strand breaks following bleomycin treatment. The authors propose here a simple system capable of detecting biological damage in DNA following irradiation doses as low as 0.5 Gy prior to bleomycin treatment. (Auth.)

Biodosimetry for medical diagnostic X-ray workers using stable chromosome aberration

International Nuclear Information System (INIS)

Wang Zhiquan; Liu Xuping; Li Jin

1996-01-01

The stable chromosome aberrations of medical diagnostic X-ray workers were analyzed using G-banding and their accumulative doses were evaluated. The results showed that the frequencies of reciprocal translocation, stable aberration and total aberration among the 4417 metaphase spread from 44 cases of medical diagnostic X-ray workers were distinctly higher than control values (P<0.05∼0.005). The stable aberration predominated strikingly in total aberration and reciprocal translocation was 57% in the stable aberrations. The medical diagnostic X-ray workers were divided into 3 groups according to calendar year of entry. The data showed that the frequencies of total aberration, stable aberration and reciprocal translocation increased with working years, especially in two groups who started working before 1970, there are statistically significant differences between the calendar year of entry before 1960 and 1960∼1969 in X-ray workers and control group. According to the equation recommended by Straume, linear coefficient (α) in linear quadratic model recommended by Schmid and the transformation coefficient by Lucas, the accumulative doses calculated are 0.58, 0.37 and 0.07 Gy for calendar year of entry before 1960, 1960∼1969 and after 1970 in X-ray workers, respectively

Stable Boundary Layer Issues

OpenAIRE

Steeneveld, G.J.

2012-01-01

Understanding and prediction of the stable atmospheric boundary layer is a challenging task. Many physical processes are relevant in the stable boundary layer, i.e. turbulence, radiation, land surface coupling, orographic turbulent and gravity wave drag, and land surface heterogeneity. The development of robust stable boundary layer parameterizations for use in NWP and climate models is hampered by the multiplicity of processes and their unknown interactions. As a result, these models suffer ...

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)

Fundamentals of the LISA stable flight formation

International Nuclear Information System (INIS)

Dhurandhar, S V; Nayak, K Rajesh; Koshti, S; Vinet, J-Y

2005-01-01

The joint NASA-ESA mission, LISA, relies crucially on the stability of the three-spacecraft constellation. Each of the spacecraft is in heliocentric orbit forming a stable triangle. In this paper we explicitly show with the help of the Clohessy-Wiltshire equations that any configuration of spacecraft lying in the planes making angles of ±60 0 with the ecliptic and given suitable initial velocities within the plane, can be made stable in the sense that the inter-spacecraft distances remain constant to first order in the dimensions of the configuration compared with the distance to the Sun. Such analysis would be useful in order to carry out theoretical studies on the optical links, simulators, etc

Wall-crossing between stable and co-stable ADHM data

Science.gov (United States)

Ohkawa, Ryo

2018-06-01

We prove formula between Nekrasov partition functions defined from stable and co-stable ADHM data for the plane following method by Nakajima and Yoshioka (Kyoto J Math 51(2):263-335, 2011) based on the theory of wall-crossing formula developed by Mochizuki (Donaldson type invariants for algebraic surfaces: transition of moduli stacks, Lecture notes in mathematics, vol 1972, Springer, Berlin, 2009). This formula is similar to conjectures by Ito et al. [J High Energy Phys 2013(5):045, 2013, (4.1), (4.2)] for A1 singularity.

Effect of CD34+ cord blood stem cell transfected by plasmid vector pIRES2-FL-IL-3 on the mice after irradiation

International Nuclear Information System (INIS)

Zhang Yong; Zhang Linsheng; Zhang Hongbing; Guo Chaohua; Tong Shiwu

2008-01-01

Objective: To observe the effect of CD34 cord blood stem cell transfected by plasmid vector plRES2-FL-IL-3 on the mouse after irradiation and to investigate its mechanisms. Methods: In the co-expressed group (12 mice), CD 34 + cord blood stem cells were transfected by plasmid vector pIRES2-FL-IL-3.5 x lO 5 cells were injected intravenously in the mouse. The hemogram changes in mice were detected 2, 4 and 6 weeks after radiation. At 6 weeks after irradiation, the expression of the CD 34 in spleen was detected by immumofluorescence method. The mRNA level and the activity of IL-3 and FL were detected by RT-PCR and Western blot. Other 3 groups were CD 34 + cell group (CD 34 group), pIRES2-IL-3 group(IL 3 group) and pIRES2-FL group(FL group), and there were 12 mice in each group. Results: The survival rate of CD 34 group, IL3 group and FL group at the 6th week were 25.0% (3/12), 50.0% (6/12) and 50.0% (6/12), respectively. It was 91.7% (11/12) in the co-expressed group, which was higher than those in the other groups. The expression of the CD 34 of spleen in the co-expressed group was higher than those of the other groups. The mRNA level and the activity of IL-3 and FL of spleen in the co-expressed group were higher than those in the other groups too. Conclusions: The CD 34 '+ cord blood stem cells transfected by plasmid vector pIRES2-FL-IL-3 have hemogenesis promotion effect on the mice after irradiation, which was related with the aggregation, proliferation of stem cells and the high expression of the interest proteins.. (authors)

Dynamics and control of twisting bi-stable structures

Science.gov (United States)

Arrieta, Andres F.; van Gemmeren, Valentin; Anderson, Aaron J.; Weaver, Paul M.

2018-02-01

Compliance-based morphing structures have the potential to offer large shape adaptation, high stiffness and low weight, while reducing complexity, friction, and scalability problems of mechanism based systems. A promising class of structure that enables these characteristics are multi-stable structures given their ability to exhibit large deflections and rotations without the expensive need for continuous actuation, with the latter only required intermittently. Furthermore, multi-stable structures exhibit inherently fast response due to the snap-through instability governing changes between stable states, enabling rapid configuration switching between the discrete number of programmed shapes of the structure. In this paper, the design and utilisation of the inherent nonlinear dynamics of bi-stable twisting I-beam structures for actuation with low strain piezoelectric materials is presented. The I-beam structure consists of three compliant components assembled into a monolithic single element, free of moving parts, and showing large deflections between two stable states. Finite element analysis is utilised to uncover the distribution of strain across the width of the flange, guiding the choice of positioning for piezoelectric actuators. In addition, the actuation authority is maximised by calculating the generalised coupling coefficient for different positions of the piezoelectric actuators. The results obtained are employed to tailor and test I-beam designs exhibiting desired large deflection between stable states, while still enabling the activation of snap-through with the low strain piezoelectric actuators. To this end, the dynamic response of the I-beams to piezoelectric excitation is investigated, revealing that resonant excitations are insufficient to dynamically trigger snap-through. A novel bang-bang control strategy, which exploits the nonlinear dynamics of the structure successfully triggers both single and constant snap-through between the stable states

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

Angina Pectoris (Stable Angina)

Science.gov (United States)

... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Angina Pectoris (Stable Angina) Updated:Aug 21,2017 You may have heard the term “angina pectoris” or “stable angina” in your doctor’s office, ...

Relevant signs of stable and unstable thoracolumbar vertebral column trauma

International Nuclear Information System (INIS)

Gehweiler, J.A.; Daffner, R.H.; Osborne, R.L.

1981-01-01

One-hundred and seventeen patients with acute thoracolumbar vertebral column fracture or fracture-dislocations were analyzed and classified into stable (36%) and unstable (64%). Eight helpful roentgen signs were observed that may serve to direct attention to serious underlying, often occult, fractures and dislocations. The changes fall into four principal groups: abnormal soft tissues, abnormal vertebral alignment, abnormal joints, and widened vertebral canal. All stable and unstable lesions showed abnormal soft tissues, while 70% demonstrated kyphosis and/or scoliosis, and an abnormal adjacent intervertebral disk space. All unstable lesions showed one or more of the following signs: displaced vertebra, widened interspinous space, abnormal apophyseal joint(s), and widened vertebral canal. (orig.)

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.

ScreenFect A: an efficient and low toxic liposome for gene delivery to mesenchymal stem cells.

Science.gov (United States)

Li, Li-Ming; Ruan, Gui-Xin; HuangFu, Ming-Yi; Chen, Zhi-Lan; Liu, Hui-Na; Li, Lin-Xian; Hu, Yu-Lan; Han, Min; Davidson, Gary; Levkin, Pavel A; Gao, Jian-Qing

2015-07-05

Mesenchymal stem cells (MSCs) hold great promise in variety of therapeutic applications including tissue engineering and cancer therapy. Genetic modification of MSCs can be used to enhance the therapeutic effect of MSCs by facilitating a specific function or by transforming MSCs into more effective gene therapy tools. However, the successful generation of genetically modified MSCs is often limited by the poor transfection efficiency or high toxicity of available transfection reagents. In our previous study, we used thiol-yne click chemistry to develop new liposomal vectors, including ScreenFect(®) A (SF) (Li et al., 2012). In this study, we investigated the transfection performance of SF on MSCs. A comparative evaluation of transfection efficiency, cell viability and cellular DNA uptake was performed using the Lipofectamine™ 2000 (L2K) as a control, and the results show that SF is superior to L2K for MSC transfection. The presence of serum did not significantly influence the transfection efficiency of either SF or L2K but greatly reduced the viability of MSC transfected by L2K. The higher efficiency of SF-mediated transfection compared to L2K was also correlated with better proliferation of cells. These results were supported by monitoring the intracellular fate of DNA, which confirmed stable transportation of DNA from lysosomes and efficient nuclear localization. TGF-β1 gene delivery by SF promoted MSC osteogenic differentiation in an osteogenic induction condition. As the first study of SF lipofection on stem cells, this study highlights a promising role of SF in gene delivery to MSCs as well as other stem cells to facilitate tissue engineering and other therapeutic effects based on genetically modified stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Normal modified stable processes

DEFF Research Database (Denmark)

Barndorff-Nielsen, Ole Eiler; Shephard, N.

2002-01-01

Gaussian (NGIG) laws. The wider framework thus established provides, in particular, for added flexibility in the modelling of the dynamics of financial time series, of importance especially as regards OU based stochastic volatility models for equities. In the special case of the tempered stable OU process......This paper discusses two classes of distributions, and stochastic processes derived from them: modified stable (MS) laws and normal modified stable (NMS) laws. This extends corresponding results for the generalised inverse Gaussian (GIG) and generalised hyperbolic (GH) or normal generalised inverse...

Preparation of thermally stable nanocrystalline hydroxyapatite by hydrothermal method.

Science.gov (United States)

Prakash Parthiban, S; Elayaraja, K; Girija, E K; Yokogawa, Y; Kesavamoorthy, R; Palanichamy, M; Asokan, K; Narayana Kalkura, S

2009-12-01

Thermally stable hydroxyapatite (HAp) was synthesized by hydrothermal method in the presence of malic acid. X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FT-IR), Raman spectroscopy, scanning electron microscopy (SEM), differential thermal analysis (DTA), thermogravimetric analysis (TGA) was done on the synthesized powders. These analyses confirmed the sample to be free from impurities and other phases of calcium phosphates, and were of rhombus morphology along with nanosized particles. IR and Raman analyses indicated the adsorption of malic acid on HAp. Thermal stability of the synthesized HAp was confirmed by DTA and TGA. The synthesized powders were thermally stable upto 1,400 degrees C and showed no phase change. The proposed method might be useful for producing thermally stable HAp which is a necessity for high temperature coating applications.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

Science.gov (United States)

Kondo, Norihiko; Ishii, Yasuyuki; Son, Aoi; Sakakura-Nishiyama, Junko; Kwon, Yong-Won; Tanito, Masaki; Nishinaka, Yumiko; Matsuo, Yoshiyuki; Nakayama, Toshinori; Taniguchi, Masaru; Yodoi, Junji

2004-03-29

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

Uses of stable isotopes

International Nuclear Information System (INIS)

Axente, Damian

1998-01-01

The most important fields of stable isotope use with examples are presented. These are: 1. Isotope dilution analysis: trace analysis, measurements of volumes and masses; 2. Stable isotopes as tracers: transport phenomena, environmental studies, agricultural research, authentication of products and objects, archaeometry, studies of reaction mechanisms, structure and function determination of complex biological entities, studies of metabolism, breath test for diagnostic; 3. Isotope equilibrium effects: measurement of equilibrium effects, investigation of equilibrium conditions, mechanism of drug action, study of natural processes, water cycle, temperature measurements; 4. Stable isotope for advanced nuclear reactors: uranium nitride with 15 N as nuclear fuel, 157 Gd for reactor control. In spite of some difficulties of stable isotope use, particularly related to the analytical techniques, which are slow and expensive, the number of papers reporting on this subject is steadily growing as well as the number of scientific meetings organized by International Isotope Section and IAEA, Gordon Conferences, and regional meeting in Germany, France, etc. Stable isotope application development on large scale is determined by improving their production technologies as well as those of labeled compound and the analytical techniques. (author)

A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides.

Science.gov (United States)

Lemaire, Géraldine; de Sousa, Georges; Rahmani, Roger

2004-12-15

A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomes improves the migratory ability of bone marrow stromal cells.

Science.gov (United States)

Wang, Gong; Zhuo, Zhongxiong; Zhang, Qian; Xu, Yali; Wu, Shengzheng; Li, Lu; Xia, Hongmei; Gao, Yunhua

2015-01-01

Bone marrow stromal cells (BMSCs) have proven useful for the treatment of various human diseases and injuries. However, their reparative capacity is limited by their poor migration and homing ability, which are primarily dependent on the SDF-1/CXCR4 axis. Most subcultured BMSCs lack CXCR4 receptor expression on the cell surface and exhibit impaired migratory capacity. To increase responsiveness to SDF-1 and promote cell migration and survival of cultured BMSCs, we used a combination of ultrasound-targeted microbubble destruction (UTMD) and liposomes to increase CXCR4 expression in vitro. We isolated and cultured rat BMSCs to their third passage and transduced them with recombinant plasmid pDsRed-CXCR4 using microbubble-mediated ultrasound irradiation and liposomes. Compared to some viral vectors, the method we employed here resulted in significantly better transfection efficiency, CXCR4 expression, and technical reproducibility. The benefits of this approach are likely due to the combination of "sonoporation" caused by shockwaves and microjet flow resulting from UTMD-generated cavitation. Following transfection, we performed a transwell migration assay and found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. The methods we describe here provide an effective, safe, non-viral means to achieve high levels of CXCR4 expression. This is associated with enhanced migration of subcultured BMSCs and may be useful for clinical application as well.

Effectiveness of Ivabradine in Treating Stable Angina Pectoris.

Science.gov (United States)

Ye, Liwen; Ke, Dazhi; Chen, Qingwei; Li, Guiqiong; Deng, Wei; Wu, Zhiqin

2016-04-01

Many studies show that ivabradine is effective for stable angina.This meta-analysis was performed to determine the effect of treatment duration and control group type on ivabradine efficacy in stable angina pectoris.Relevant articles in the English language in the PUBMED and EMBASE databases and related websites were identified by using the search terms "ivabradine," "angina," "randomized controlled trials," and "Iva." The final search date was November 2, 2015.Articles were included if they were published randomized controlled trials that related to ivabradine treatment of stable angina pectoris.Patients with stable angina pectoris were included.The patients were classified according to treatment duration (Angina outcomes were heart rate at rest or peak, exercise duration, and time to angina onset.Seven articles were selected. There were 3747 patients: 2100 and 1647 were in the ivabradine and control groups, respectively. The ivabradine group had significantly longer exercise duration when they had been treated for at least 3 months, but not when treatment time was less than 3 months. Ivabradine significantly improved time to angina onset regardless of treatment duration. Control group type did not influence the effect of exercise duration (significant) or time to angina onset (significant).Compared with beta-blocker and placebo, ivabradine improved exercise duration and time to onset of angina in patients with stable angina. However, its ability to improve exercise duration only became significant after at least 3 months of treatment.

On the joint statistics of stable random processes

International Nuclear Information System (INIS)

Hopcraft, K I; Jakeman, E

2011-01-01

A utilitarian continuous bi-variate random process whose first-order probability density function is a stable random variable is constructed. Results paralleling some of those familiar from the theory of Gaussian noise are derived. In addition to the joint-probability density for the process, these include fractional moments and structure functions. Although the correlation functions for stable processes other than Gaussian do not exist, we show that there is coherence between values adopted by the process at different times, which identifies a characteristic evolution with time. The distribution of the derivative of the process, and the joint-density function of the value of the process and its derivative measured at the same time are evaluated. These enable properties to be calculated analytically such as level crossing statistics and those related to the random telegraph wave. When the stable process is fractal, the proportion of time it spends at zero is finite and some properties of this quantity are evaluated, an optical interpretation for which is provided. (paper)

Stable isotopes labelled compounds

International Nuclear Information System (INIS)

1982-09-01

The catalogue on stable isotopes labelled compounds offers deuterium, nitrogen-15, and multiply labelled compounds. It includes: (1) conditions of sale and delivery, (2) the application of stable isotopes, (3) technical information, (4) product specifications, and (5) the complete delivery programme

Stable Boundary Layer Issues

NARCIS (Netherlands)

Steeneveld, G.J.

2012-01-01

Understanding and prediction of the stable atmospheric boundary layer is a challenging task. Many physical processes are relevant in the stable boundary layer, i.e. turbulence, radiation, land surface coupling, orographic turbulent and gravity wave drag, and land surface heterogeneity. The

The Nature of Stable Insomnia Phenotypes

Science.gov (United States)

Pillai, Vivek; Roth, Thomas; Drake, Christopher L.

2015-01-01

Study Objectives: We examined the 1-y stability of four insomnia symptom profiles: sleep onset insomnia; sleep maintenance insomnia; combined onset and maintenance insomnia; and neither criterion (i.e., insomnia cases that do not meet quantitative thresholds for onset or maintenance problems). Insomnia cases that exhibited the same symptom profile over a 1-y period were considered to be phenotypes, and were compared in terms of clinical and demographic characteristics. Design: Longitudinal. Setting: Urban, community-based. Participants: Nine hundred fifty-four adults with Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition based current insomnia (46.6 ± 12.6 y; 69.4% female). Interventions: None. Measurements and results: At baseline, participants were divided into four symptom profile groups based on quantitative criteria. Follow-up assessment 1 y later revealed that approximately 60% of participants retained the same symptom profile, and were hence judged to be phenotypes. Stability varied significantly by phenotype, such that sleep onset insomnia (SOI) was the least stable (42%), whereas combined insomnia (CI) was the most stable (69%). Baseline symptom groups (cross-sectionally defined) differed significantly across various clinical indices, including daytime impairment, depression, and anxiety. Importantly, however, a comparison of stable phenotypes (longitudinally defined) did not reveal any differences in impairment or comorbid psychopathology. Another interesting finding was that whereas all other insomnia phenotypes showed evidence of an elevated wake drive both at night and during the day, the “neither criterion” phenotype did not; this latter phenotype exhibited significantly higher daytime sleepiness despite subthreshold onset and maintenance difficulties. Conclusions: By adopting a stringent, stability-based definition, this study offers timely and important data on the longitudinal trajectory of specific insomnia phenotypes. With

Calcium-microRNA Complexes Functionalized Nanotubular Implant Surface for Highly Efficient Transfection and Enhanced Osteogenesis of Mesenchymal Stem Cells

DEFF Research Database (Denmark)

Song, Wen; Yang, Chuanxu; Svend Le, Dang Quang

2018-01-01

Controlling mesenchymal stem cells (MSCs) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible and highly...... effective delivery method of small RNA therapeutics into hMSCs from an implant surface by calcium ions. First, we demonstrated that simple Ca/siGFP nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca2+ concentration (>5 mM). In addition, a single transfection could...

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study.

Science.gov (United States)

Wang, Ruoqing; Zhang, Nianwei; Wang, Renkai; Wang, Shengpeng; Wang, Na

2017-12-01

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support.

Science.gov (United States)

Giri, Shibashish; Bader, Augustinus

2014-09-01

Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a

Estrogen receptor α and aryl hydrocarbon receptor cross-talk in a transfected hepatoma cell line (HepG2 exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Directory of Open Access Journals (Sweden)

Manuela Göttel

2014-01-01

Full Text Available The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2 in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.

Structure of acid-stable carmine.

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Sugimoto, Naoki; Kawasaki, Yoko; Sato, Kyoko; Aoki, Hiromitsu; Ichi, Takahito; Koda, Takatoshi; Yamazaki, Takeshi; Maitani, Tamio

2002-02-01

Acid-stable carmine has recently been distributed in the U.S. market because of its good acid stability, but it is not permitted in Japan. We analyzed and determined the structure of the major pigment in acid-stable carmine, in order to establish an analytical method for it. Carminic acid was transformed into a different type of pigment, named acid-stable carmine, through amination when heated in ammonia solution. The features of the structure were clarified using a model compound, purpurin, in which the orientation of hydroxyl groups on the A ring of the anthraquinone skeleton is the same as that of carminic acid. By spectroscopic means and the synthesis of acid-stable carmine and purpurin derivatives, the structure of the major pigment in acid-stable carmine was established as 4-aminocarminic acid, a novel compound.

Synthesis and Evaluation of Tetramethylguanidinium-Polyethylenimine Polymers as Efficient Gene Delivery Vectors

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Manohar Mahato

2014-01-01

Full Text Available Previously, we demonstrated that 6-(N,N,N′,N′-tetramethylguanidinium chloride-hexanoyl-polyethylenimine (THP polymers exhibited significantly enhanced transfection efficiency and cell viability. Here, in the present study, we have synthesized a series of N,N,N′,N′-tetramethylguanidinium-polyethylenimine (TP1-TP5 polymers via a single-step reaction involving peripheral primary amines of bPEI and varying amounts of 2-(1H-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU. These polymers were found to interact efficiently with negatively charged pDNA and formed stable complexes in the size range of ~240–450 nm. Acid-base titration profiles revealed improved buffering capacity of TP polymers as compared to bPEI. Transfection and cytotoxicity assays performed with TP/pDNA complexes on HEK293, CHO, and HeLa cells showed significantly higher transfection efficiency and cell viability with one of the complexes, TP2/pDNA complex, exhibited the highest transfection efficiency (~1.4–2.3-fold outcompeting native bPEI and the commercially available transfection reagent, Lipofectamine 2000. Compared to previously reported THP polymers, the transfection efficiency of TP/pDNA complexes was found to be lower, as examined by flow cytometry. These results highlight the importance of the hydrophobic C-6 linker in THP polymers in forming compact nanostructures with pDNA, which might lead to efficient uptake and internalization of the complexes; however, the projected TP polymers offer an advantage of their rapid and economical one-step synthesis.

Mitotic evolution of Plasmodium falciparum shows a stable core genome but recombination in antigen families.

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Selina E R Bopp

Full Text Available Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone. In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1 on chromosome 1. We observed 18 large-scale (>1 kb on average deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6 structural variants per base pair per generation. Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9 mutations per base pair per generation, we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum

Mitotic Evolution of Plasmodium falciparum Shows a Stable Core Genome but Recombination in Antigen Families

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Bopp, Selina E. R.; Manary, Micah J.; Bright, A. Taylor; Johnston, Geoffrey L.; Dharia, Neekesh V.; Luna, Fabio L.; McCormack, Susan; Plouffe, David; McNamara, Case W.; Walker, John R.; Fidock, David A.; Denchi, Eros Lazzerini; Winzeler, Elizabeth A.

2013-01-01

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to

Applications of stable isotopes

International Nuclear Information System (INIS)

Letolle, R.; Mariotti, A.; Bariac, T.

1991-06-01

This report reviews the historical background and the properties of stable isotopes, the methods used for their measurement (mass spectrometry and others), the present technics for isotope enrichment and separation, and at last the various present and foreseeable application (in nuclear energy, physical and chemical research, materials industry and research; tracing in industrial, medical and agronomical tests; the use of natural isotope variations for environmental studies, agronomy, natural resources appraising: water, minerals, energy). Some new possibilities in the use of stable isotope are offered. A last chapter gives the present state and forecast development of stable isotope uses in France and Europe

Palmitic acid/polypyrrole composites as form-stable phase change materials for thermal energy storage

International Nuclear Information System (INIS)

Silakhori, Mahyar; Metselaar, Hendrik Simon Cornelis; Mahlia, Teuku Meurah Indra; Fauzi, Hadi; Baradaran, Saeid; Naghavi, Mohammad Sajad

2014-01-01

Highlights: • A novel phase change composite of palmitic acid–polypyrrole(PA–PPy) was fabricated. • Thermal properties of PA–PPy are characterized in different mass ratios of PA–PPy. • Thermal cycling test showed that form stable PCM had a favorable thermal reliability. - Abstract: In this study a novel palmitic acid (PA)/polypyrrole (PPy) form-stable PCMs were readily prepared by in situ polymerization method. PA was used as thermal energy storage material and PPy was operated as supporting material. Form-stable PCMs were investigated by SEM (scanning electron microscopy) and FTIR (Fourier transform infrared spectrometer) analysis that illustrated PA Particles were wrapped by PPy particles. XRD (X-ray diffractometer) was used for crystalline phase of PA/PPy composites. Thermogravimetry analysis (TGA) and differential scanning calorimetry (DSC) were used for investigating Thermal stability and thermal energy storage properties of prepared form-stable PCMs. According to the obtained results the form stable PCMs exhibited favorable thermal stability in terms of their phase change temperature. The form-stable PCMs (79.9 wt% loading of PA) were considered as the highest loading PCM with desirable latent heat storage of 166.3 J/g and good thermal stability. Accelerated thermal cycling tests also showed that form stable PCM had an acceptable thermal reliability. As a consequence of acceptable thermal properties, thermal stability and chemical stability, we can consider the new kind of form stable PCMs for low temperature solar thermal energy storage applications

Radiostatine and radioiodine uptake characterization in sodium iodine symporter-expressing cell lines

International Nuclear Information System (INIS)

Petrich, T.; Helmeke, H.J.; Meyer, G.J.; Knapp, W.H.; Poetter, E.

2002-01-01

Full text: The sodium iodide symporter (NIS) has been recognized as an attractive target for cancer gene therapy. Here we investigated NIS-mediated transport of the high LET α-emitter astatine, 211 At, in comparison to radioiodine. A constitutive expression vector harbouring the human NIS cDNA was used in combination with reporter gene vectors for transient transfection of 13 different human cancer cell lines. Radioiodine uptake was measured as well as transfection efficiencies. Six stable NIS-expressing cell lines (3 derived from thyroid carcinomas, 2 colon carcinoma, 1 glioblastoma) were generated by antibiotic selection. NIS expression was monitored by immunohistochemistry and RT-PCR. Subsequently the radioastatine and radioiodine uptake characteristics of genetically modified cells were studied in comparison to the respective control cells. After xenotransplantation in nude mice in vivo tumor imaging by scintigraphy and biodistribution studies following organ removal were performed. Transient transfection of NIS cDNA led to high specific sodium perchlorate-sensitive radioiodine uptake in NIS-expressing cells that roughly correlates to transfection efficiencies. Similarly, stable NIS-expressing cell lines were able to concentrate high levels of radioiodine and in addition showed comparable transport capacity for radioastatine. Accumulation of 211 At was inhibited by sodium perchlorate like iodide uptake and displayed dependency an extracellular Na + - and I - -ions as well. Compared to wash-out experiments in cell culture the effective half life of radioiodine and radioastatine in vivo was significantly prolonged. Preliminary dose calculations by MIRD concepts indicated higher tumor radiation doses for 211 At compared to 131 I. Tumor cells of different origins transfected with the NIS-expression vector specifically and significantly take-up radioiodine and radioastatine in vitro and in vivo. The data provide direct evidence that the NIS efficiently transports

Comprehensive first-principles study of stable stacking faults in hcp metals

International Nuclear Information System (INIS)

Yin, Binglun; Wu, Zhaoxuan; Curtin, W.A.

2017-01-01

The plastic deformation in hcp metals is complex, with the associated dislocation core structures and properties not well understood on many slip planes in most hcp metals. A first step in establishing the dislocation properties is to examine the stable stacking fault energy and its structure on relevant slip planes. However, this has been perplexing in the hcp structure due to additional in-plane displacements on both sides of the slip plane. Here, density functional theory guided by crystal symmetry analysis is used to study all relevant stable stacking faults in 6 hcp metals (Mg, Ti, Zr, Re, Zn, Cd). Specially, the stable stacking fault energy, position, and structure on the Basal, Prism I and II, Pyramidal I and II planes are determined using all-periodic supercells with full atomic relaxation. All metals show similar stacking fault position and structure as dictated by crystal symmetry, but the associated stacking fault energy, being governed by the atomic bonding, differs significantly among them. Stacking faults on all the slip planes except the Basal plane show substantial out-of-plane displacements while stacking faults on the Prism II, Pyramidal I and II planes show additional in-plane displacements, all extending to multiple atom layers. The in-plane displacements are not captured in the standard computational approach for stacking faults, and significant differences are shown in the energies of such stacking faults between the standard approach and fully-relaxed case. The existence of well-defined stable stacking fault on the Pyramidal planes suggests zonal dislocations are unlikely. Calculations on the equilibrium partial separation further suggests 〈c + a〉 dissociation into three partials on the Pyramidal I plane is unlikely and 〈c〉 dissociation on Prism planes is unlikely to be stable against climb-dissociation onto the Basal planes in these metals.

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

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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.

Stable isotope composition of human fingernails from Slovakia

International Nuclear Information System (INIS)

Grolmusová, Zuzana; Rapčanová, Anna; Michalko, Juraj; Čech, Peter; Veis, Pavel

2014-01-01

Stable isotope composition of human fingernails has proven to be useful for documenting human dietary information and geographical patterns in archeological, forensic, anthropological and biological studies. Therefore, it is of interest to detect all factors influencing the stable isotopic composition in the certain regions in the world. Carbon and nitrogen isotope data of human fingernail keratin from 52 individuals from Slovakia were reported in this study. The online combustion and continuous flow isotope-ratio mass spectrometer Delta V Advantage was used for δ 13 C and δ 15 N analysis of fingernail keratin samples from 24 vegetarian and 28 omnivorous individuals. A group of people with frequent meat consumption showed enrichment in 13 C and 15 N isotopes in fingernails. A similar trend was observed with increasing seafood in an individual's diet. Moreover a significant difference was revealed between smokers and nonsmokers for both δ 13 C and δ 15 N values. These data were compared to previously published δ 13 C and δ 15 N fingernail values from across the globe. This study brings new information on the stable isotope signature of individuals from Slovakia and characterizes the Central European region for the first time. The stable isotope composition of fingernails is influenced by the frequency of meat and seafood consumption as well as smoking. - Highlights: • This study deals with stable isotope analyses of fingernails from Slovak volunteers. • δ 13 C and δ 15 N values of vegetarian and omnivore fingernails were compared. • Influence of sex, diet and smoking was studied

Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

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Wang, Fei; Wang, Yitong; Wang, Hui; Shao, Naimin; Chen, Yuanyuan; Cheng, Yiyun

2014-11-01

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Efficient and nontoxic biological response carrier delivering TNF-α shRNA for gene silencing in a murine model of rheumatoid arthritis

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Jialin Song

2016-08-01

Full Text Available Small interfering RNA (siRNA is an effective and specific method for silencing genes. However, an efficient and nontoxic carrier is needed to deliver the siRNA into the target cells. Tumor necrosis factor α (TNF-α plays a central role in the occurrence and progression of rheumatoid arthritis. In this study, we pre-synthetized a degradable cationic polymer (PDAPEI from 2,6-pyridinedicarboxaldehyde and low molecular weight polyethyleneimine (PEI, Mw=1.8 kDa as a gene vector for the delivery of TNF-α shRNA. The PDAPEI/pDNA complex showed a suitable particle size and stable zeta potential for transfection. In vitro study of the PDAPEI/pDNA complex revealed a lower cytotoxicity and higher transfection efficiency when transfecting TNF-α shRNA to macrophages by significantly down-regulating the expression of TNF-α. Moreover, the complex was extremely efficient in decreasing the severity of arthritis in mice with collagen-induced arthritis (CIA. PDAPEI delivered TNF-α shRNA has great potential in the treatment of rheumatoid arthritis.

Looking for chemical reaction networks exhibiting a drift along a manifold of marginally stable states.

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Brogioli, Doriano

2013-02-07

I recently reported some examples of mass-action equations that have a continuous manifold of marginally stable stationary states [Brogioli, D., 2010. Marginally stable chemical systems as precursors of life. Phys. Rev. Lett. 105, 058102; Brogioli, D., 2011. Marginal stability in chemical systems and its relevance in the origin of life. Phys. Rev. E 84, 031931]. The corresponding chemical reaction networks show nonclassical effects, i.e. a violation of the mass-action equations, under the effect of the concentration fluctuations: the chemical system drifts along the marginally stable states. I proposed that this effect is potentially involved in abiogenesis. In the present paper, I analyze the mathematical properties of mass-action equations of marginally stable chemical reaction networks. The marginal stability implies that the mass-action equations obey some conservation law; I show that the mathematical properties of the conserved quantity characterize the motion along the marginally stable stationary state manifold, i.e. they allow to predict if the fluctuations give rise to a random walk or a drift under the effect of concentration fluctuations. Moreover, I show that the presence of the drift along the manifold of marginally stable stationary-states is a critical property, i.e. at least one of the reaction constants must be fine tuned in order to obtain the drift. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of Rad 51 overexpression on chromosomal stability and radiation sensitivity in tumour cells

International Nuclear Information System (INIS)

Jend, C.; Stuerzbecher, H.W.; Dikomey, E.; Borgmann, K.

2004-01-01

The present study was dedicated to examining the effects of Rad51 overexpression on genomic instability, expressed in terms of chromosomal aberrations in G1 and G2 phases following X-ray irradiation. For this purpose an osteosarcoma cell line (Ui-OS) which shows inducing Rad51 overexpression (UiRad5-2) after stable transfection was compared with an isogenetic line (UiLacZ) which overexpresses beta-galactosidase instead of Rad51 [de

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue