Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI™ and Lipofectamine™ 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI™ was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.
Kim, T.K. and Eberwine, J.H., Mammalian Cell Transfection: the Present and the Future, Anal. Bioanal. Chem., 2010, vol. 397, pp. 3173–3178.
Recillas-Targa, F., Multiple Strategies for Gene Transfer, Expression, Knockdown, and Chromatin Influence in Mammalian Cell Lines and Transgenic Animals, Mol. Biotechnol., 2006, vol. 34, pp. 337–354.
Shabani, M., Hemmati, Sh., Hadavi, R., et al., Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents, Avicenna, J. Med. Biotech., 2010, vol. 2, pp. 123–130.
Maurisse, R., Semir, D.D., Emamekhoo, H., et al., Comparative Transfection of DNA into Primary and Transformed Mammalian Cells from Different Lineages, BMC Biotechnol., 2010, vol. 10, pp. 2–9.
Colosimo, A., Goncz, K.K., Holmes, A.R., et al., Transfer and Expression of Foreign Genes in Mammalian Cells, BioTechniques, 2000, vol. 29, pp. 314–331.
Heiser, W.C., Optimizing Electroporation Conditions for the Transformation of Mammalian Cells, in Methods in Molecular Biology, Transcription Factor Protocols, Tymms M.J., Ed., Humana Press, 2000, vol. 130, pp. 117–134.
Melkonyan, H., Sorg, C., and Klempt, M., Electroporation Efficiency in Mammalian Cells Is Increased by Dimethyl Sulfoxide (DMSO), Nucleic Acids Res., 1996, vol. 24, pp. 4356–4357.
Vecchi, C., Montosi, G., and Pietrangelo, A., Huh 7: A Human “Hemochromatotic” Cell Line, Hepatology, 2010, vol. 51, pp. 654–659.
Lin, W., Choe, W.H., Hiasa, Y., et al., Hepatitis C Virus Expression Suppresses Interferon Signaling by Degrading STAT1, Gastroenterology, 2005, vol. 128, pp. 1034–1041.
Ciccaglione, A.R., Stellacci, E., Marcantonio, C., et al., Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes, J. Virol., 2007, vol. 81, pp. 202–214.
Evans, S., Cavanagh, D., and Britton, P., Utilizing Fowl-Pox Virus Recombinants to Generate Defective RNAs of the Coronavirus Infectious Bronchitis Virus, J. Gen. Virol., 2000, vol. 81, pp. 2855–2865.
Kistner, O., Howard, K., Spruth, M., et al., Cell Culture (Vero) Derived Whole Virus (H5N1) Vaccine Based on Wild-Type Virus Strain Induces Cross-Protective Immune Responses, Vaccine, 2007, vol. 25, pp. 6028–6036.
Michel, M.R., Elgizoli, M., Dai, Y., et al., Karyophilic Properties of Semliki Forest Virus Nucleocapsid Protein, J. Virol., 1990, vol. 64, pp. 5123–5131.
Cao, F., Xie, X., Gollan, T., et al., Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells, Mol. Imaging Biol, 2010, vol. 12, pp. 15–24.
Lakshmipathy, U., Pelacho, B., Sudo, K., et al., Efficient Transfection of Embryonic and Adult Stem Cells, Stem Cells, 2004, vol. 22, pp. 531–543.
Engler, C., Kelliher, C., Wahlin, K.J., Speck, C.L., and Jun, A.S., Comparison of Non-Viral Methods to Genetically Modify and Enrich Populations of Primary Human Corneal Endothelial Cells, Mol. Vis., 2009, vol. 15, pp. 629–637.
Chen, L., Sun, J., Meng, L., et al., ISG15, a Ubiquitin-Like Interferon-Stimulated Gene, Promotes Hepatitis C Virus Production in vitro: Implications for Chronic Infection and Response to Treatment, J. Gen. Virol., 2010, vol. 91, pp. 382–388.
Melen, K., Fagerlund, R., Nyqvist, M., et al., Expression of Hepatitis C Virus Core Protein Inhibits Interferon Induced Nuclear Import of STATs, J. Med. Virol., 2004, vol. 73, pp. 536–547.
Mello, F.C.A., Martel, N., Gomes, S.A., and Araujo, N.M., Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection, Hepat. Res. Treat., 2011, vol. 2011, pp. 695–859.
Beare, P.A., Howe, D., Cockrell, D.C., et al., Characterization of a Coxiella burnetii FtsZ Mutant Generated by Himar1 Transposon Mutagenesis, J. Bacteriol., 2009, vol. 191, pp. 1369–1381.
Saffran, H.A., Read, G.S., and Smiley, J.R., Evidence for Translational Regulation by the Herpes Simplex Virus Virion Host Shutoff Protein, J. Virol., 2010, vol. 84, pp. 6041–6049.
Gray, W.L., Zhou, F., Noffke, J., and Tischer, B.K., Cloning the Simian Varicella Virus Genome in E. coli as an Infectious Bacterial Artificial Chromosome, Arch. Virol., 2011, pp. 1–8.
Gonzalez, G., Pfannesa, L., Brazas, R., and Strikera, R., Selection of an Optimal RNA Transfection Reagent and Comparison to Electroporation for the Delivery of Viral RNA, J. Virol. Methods, 2007, vol. 145, pp. 14–21.
Uchida, E., Mizuguchi, H., Ishiiwatabe, A., and Hayakawa, T., Comparison of the Efficiency and Safety of Non-Viral Vector-Mediated Gene Transfer Into a Wide Range of Human Cells, Biol. Pharm. Bull., 2002, vol. 25, pp. 891–897.
Schwartz, B., Ivanov, M.A., Pitard, B., et al., Synthetic DNA-Compacting Peptides Derived from Human Sequence Enhance Cationic Lipid-Mediated Gene Transfer in vitro and in vivo, Gene Ther., 1999, vol. 6, pp. 282–292.
Kirkham, M. and Parton, R.G., Clathrin-Independent Endocytosis: New Insights Into Caveolae and Noncaveolar Lipid Raft Carriers, Bba-Mol, Cell Res., 2005, vol. 1746, pp. 350–363.
Vercauteren, D., Vandenbroucke, R.E., Jones, A.T., et al., The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls, Mol. Ther., 2009, vol. 18, pp. 561–569.
Mennesson, E., Fuchs, R., et al., Mac-Ropinocytosis of Polyplexes and Recycling of Plasmid via the Clathrin-Dependent Pathway Impair the Transfection Efficiency of Human Hepatocarcinoma Cells, Mol. Ther., 2004, vol. 10, pp. 373–385.
Rejman, J., Bragonzi, A., and Conese, M., Role of Clathrin-and Caveolae-Mediated Endocytosis in Gene Transfer Mediated by Lipo- and Polyplexes, Mol. Ther., 2005, vol. 12, pp. 468–474.
Von Gersdorff, K., Sanders, N.N., Vandenbroucke, R., et al., The Internalization Route Resulting in Successful Gene Expression Depends on Both Cell Line and Polyethylenimine Polyplex Type, Mol. Ther., 2006, vol. 14, pp. 745–753.
Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P.H., Gene Transfer Into Mouse Lyoma Cells by Electroporation in High Electric Fields, EMBO J., 1982, vol. 1, pp. 841–842.
Golub, E.I., Kim, H., and Volsky, D.J., Transfection of DNA Iinto Adherent Cells by DEAE-Dextran/DMSO Method Increases Drastically If the Cells Are Removed from Surface and Treated in Suspension, Nucleic Acids Res., 1989, vol. 17, p. 4902.
Chisholm, O. and Symonds, G., Transfection of Myeloid Cell Lines Using Polybrene/DMSO, Nucleic Acids Res., 1988, vol. 16, p. 2352.
Kawai, S. and Nishizawa, M., New Procedure for DNA Transfection with Polycation and Dimethyl Sulfoxide, Mol. Cell Biol., 1984, vol. 4, p. 1172.
Sambrook, J. and Russell, D.W., DNA Transfection by Electroporation, in Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, vol. 1, p. 16.33–16.36.
Potter, H. and Heller, R., Transfection by Electroporation, Curr. Protoc. Mol. Biol., Wiley, 2003, pp. 9.3.1–9.3.6.
Bergan, R., Connell, Y., Fahmy, B., and Neckers, L., Electroporation Enhances C-Myc Antisense Oligodeoxy-Nucleotide Efficacy, Nucleic Acids Res., 1993, vol. 21, pp. 3567–3573.
The article is published in the original.
About this article
Cite this article
Hashemi, A., Roohvand, F., Ghahremani, M.H. et al. Optimization of transfection methods for Huh-7 and Vero cells: A comparative study. Cytol. Genet. 46, 347–353 (2012). https://doi.org/10.3103/S0095452712060035
- Transfection Efficiency
- Electric Field Strength
- Vero Cell
- Infectious Bronchitis Virus
- Cationic Lipid