Molecular Biotechnology

, Volume 34, Issue 2, pp 225–237 | Cite as

Large-Scale transfection of mammalian cells for the fast production of recombinant protein



Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.

Index Entries

HEK 293 CHO COS EBNA1 polyethylenimine suspension serum-free gene transfer 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Graham, F. L. and van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467.PubMedCrossRefGoogle Scholar
  2. 2.
    Blasey, H. D. and Bernard, A. R. (1994) Transient expression with COS cells on spinner scale, In Animal Cell Technology: Products of Today, Prospects for Tomorrow, (Spier, R. E., Griffiths, J. B., and Berthold, W., eds.) Oxford, UK, pp. 331–332.Google Scholar
  3. 3.
    Blasey, H. D., Aubry, J. P., Mazzei, G. J., and Bernard, A. R. (1996) Large scale transient expression with COS cells. Cytotechnology, 18, 183–192.CrossRefGoogle Scholar
  4. 4.
    Blasey, H. D., Hovius, R., Vogel, H., and Bernard, A. R. (1999) Transient-expression technologies, their application and scale-up: 5-HT3 serotonin receptor case study. Biochem. Soc. Trans. 27, 956–960.PubMedGoogle Scholar
  5. 5.
    Geisse, S., Gram, H., Kleuser, B., and Kocher, H. P. (1996) Eukaryotic expression systems: a comparison. Protein Expr. Purif. 8, 271–282.PubMedCrossRefGoogle Scholar
  6. 6.
    Geisse, S. and Kocher, H. P. (1999) Protein expression in mammalian and insect cell systems. Meth. Enzymol. 306, 19–42.PubMedGoogle Scholar
  7. 7.
    Ridder, R., Geisse, S., Kleuser, B., Kawalleck, P., and Gram, H. (1995) A COS-cell-based system for rapid production and quantification of scVv::IgC kappa antibody fragments. Gene 166, 273–276.PubMedCrossRefGoogle Scholar
  8. 8.
    Jordan, M., Köhne, C., and Wurm, F. M. (1998) Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotechnology 26, 39–47.CrossRefGoogle Scholar
  9. 9.
    Schlaeger, E. J., Legendre, J. Y., Trzeciak, A., et al. (1998) Transient transfection in mammalian cells: a basic study for an efficient and cost-effective scale up process. In: New Developments and New Applications in Animal Cell Technology: Proceedings of the 15th ESACT Meeting (Merten, O. W., Perrin, P., and Griffiths, B., eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 105–112.Google Scholar
  10. 10.
    Schlaeger, E.-J. and Christensen, K. (1999) Transient gene expression in mammalian cells grown in serumfree suspension culture. Cytotechnology 30, 71–83.CrossRefGoogle Scholar
  11. 11.
    Wurm, F. and Bernard, A. (1999) Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10, 156–159.PubMedCrossRefGoogle Scholar
  12. 12.
    Capecchi, M. R. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488.PubMedCrossRefGoogle Scholar
  13. 13.
    Graessmann, M., Menne, J., Liebler, M., Graeber, I., and Graessmann, A. (1989) Helper activity for gene expression, a novel function of the SV40 enhancer. Nucleic Acids Res. 17, 6603–6612.PubMedCrossRefGoogle Scholar
  14. 14.
    Colosimo, A., Goncz, K. K., Holmes, A. R., et al. (2000) Transfer and expression of foreign genes in mammalian cells. Biotechniques 29, 314–324.PubMedGoogle Scholar
  15. 15.
    Schenborn, E. T. and Oler, J. (2000) Liposome-mediated transfection of mammalian cells. Methods Mol. Biol. 130, 155–164.PubMedGoogle Scholar
  16. 16.
    Haberland, A. and Bottger, M. (2005) Nuclear proteins as gene-transfer vectors. Biotechnol. Appl. Biochem. 42, 97–106.PubMedCrossRefGoogle Scholar
  17. 17.
    Li, L. H., Shivakumar, R., Feller, S., et al. (2002) Highly efficient, large volume flow electroporation. Technol. Cancer Res. Treat. 1, 341–350.PubMedGoogle Scholar
  18. 18.
    Baldi, L., Muller, N., Picasso, S., et al. (2005) Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol. Prog. 21, 148–153.PubMedCrossRefGoogle Scholar
  19. 19.
    Derouazi, M., Girard, P., Van, F. T., et al. (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng. 87, 537–545.PubMedCrossRefGoogle Scholar
  20. 20.
    Durocher, Y., Perret, S., and Kamen, A. (2002) High-level and high-throughput recombinant protein proeuction by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, E9.PubMedCrossRefGoogle Scholar
  21. 21.
    Geisse, S., and Henke, M. (2005) Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J. Struct. Funct. Genomics 6, 165–170.PubMedCrossRefGoogle Scholar
  22. 22.
    Girard, P., Jordan, M., Tsao, M., and Wurm, F. M. (2001) Small-scale bioreactor system for process development and optimization. Biochem. Eng. J. 7, 117–119.PubMedCrossRefGoogle Scholar
  23. 23.
    Girard, P., Derouazi, M., Baumgartner, G., et al. (2002) 100-liter transient transfection. Cytotechnology 38, 15–21.CrossRefGoogle Scholar
  24. 24.
    Meissner, P., Pick, H., Kulangara, A., Chatellard, P., Friedrich, K., and Wurm, F. M. (2001) Transient gene expression: recombinant protein production with suspension-adapted HEK-293-EBNA cells. Biotechnol. Bioeng. 75, 197–203.PubMedCrossRefGoogle Scholar
  25. 25.
    Pham, P. L., Perret, S., Doan, H. C., et al. (2003) Large-scale transient transfection of serum-free suspension-growing HEK-293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol. Bioeng. 84, 332–342.PubMedCrossRefGoogle Scholar
  26. 26.
    Schlaeger, E.-J., Kitas, E. A., and Dorn, A. (2003) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture. Cytotechnology 42, 47–55.CrossRefGoogle Scholar
  27. 27.
    Pham, P. L., Perret, S., Cass, B., et al. (2005) Transient gene expression in HEK293 cells: peptone addition posttranfection improves recombinant protein synthesis. Biotechnol. Bioeng. 90, 332–344.PubMedCrossRefGoogle Scholar
  28. 28.
    Haldankar, R., Li, G., Zane, S., Baikalov, C., and Deshpande, R. (2006) Serum-free suspension largescale transient transfection of CHO cells in WAVE bioreactors. Mol. Biotechnol., this issue.Google Scholar
  29. 29.
    Jordan, M. and Wurm, F. (2004) Transfection of adherent and suspended cells by calcium phosphate. Methods 33, 136–143.PubMedCrossRefGoogle Scholar
  30. 30.
    Loyter, A., Scangos, G., Juricek, D., Keene, D., and Ruddle, F. H. (1982) Mechanisms of DNA entry into mammalian cells. II. Phagocytosis of calcium phosphate DNA co-precipitate visualized by electron microscopy. Exp. Cell Res. 139, 223–234.PubMedCrossRefGoogle Scholar
  31. 31.
    Orrantia, E. and Chang, P. L. (1990) Intracellular distribution of DNA internalized through calcium phosphate precipitation. Exp. Cell Res. 190, 170–174.PubMedCrossRefGoogle Scholar
  32. 32.
    Orrantia, E., Li, Z. G., and Chang, P. L. (1990) Energy dependence of DNA-mediated gene transfer and expression. Somat. Cell Mol. Genet. 16, 305–310.PubMedCrossRefGoogle Scholar
  33. 33.
    Coonrod, A., Li, F. Q., and Horwitz, M. (1997) On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther. 4, 1313–1321.PubMedCrossRefGoogle Scholar
  34. 34.
    Loyter, A., Scangos, G. A., and Ruddle, F. H. (1982) Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc. Natl. Acad. Sci. USA 79, 422–426.PubMedCrossRefGoogle Scholar
  35. 35.
    Jordan, M., Schallhorn, A., and Wurm, F. M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601.PubMedCrossRefGoogle Scholar
  36. 36.
    Batard, P., Jordan, M., and Wurm, F. (2001) Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270, 61–68.PubMedCrossRefGoogle Scholar
  37. 37.
    Atkinson, A. and Jack, G. W. (1973) Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids and proteins on immobilised polyethyleneimine. Biochim. Biophys. Acta 308, 41–52.PubMedGoogle Scholar
  38. 38.
    Boussif, O., Lezoualc'h, F., Zanta, M. A., et al. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA, 92, 7297–7301.PubMedCrossRefGoogle Scholar
  39. 39.
    Lungwitz, U., Breunig, M., Blunk, T., and Gopferich, A. (2005) Polyethylenimine-based non-viral gene delivery systems. Eur. J. Pharm. Biopharm. 60, 247–266.PubMedCrossRefGoogle Scholar
  40. 40.
    Neu, M., Fischer, D., and Kissel, T. (2005) Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J. Gene Med. 7, 992–1009.PubMedCrossRefGoogle Scholar
  41. 41.
    Mislick, K. A. and Baldeschwieler, J. D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. USA 93, 12,349–12,354.CrossRefGoogle Scholar
  42. 42.
    Bieber, T., Meissner, W., Kostin, S., Niemann, A., and Elsasser, H. P. (2002) Intracellular route and transcriptional competence of polyethylenimine-DNA complexes. J. Control Release 82, 441–454.PubMedCrossRefGoogle Scholar
  43. 43.
    Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. USA 96, 5177–5181.PubMedCrossRefGoogle Scholar
  44. 44.
    Remy-Kristensen, A., Clamme, J. P., Vuilleumier, C., Kuhry, J. G., and Mely, Y. (2001) Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim. Biophys. Acta 1514, 21–32.PubMedCrossRefGoogle Scholar
  45. 45.
    Akinc, A., Thomas, M., Klibanov, A. M., and Langer, R. (2005) Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J. Gene Med. 7, 657–663.PubMedCrossRefGoogle Scholar
  46. 46.
    Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., and Escande, D. (1998) Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem. 273, 7507–7511.PubMedCrossRefGoogle Scholar
  47. 47.
    Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. (2000) Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275, 1625–1629.PubMedCrossRefGoogle Scholar
  48. 48.
    Lechardeur, D., Sohn, K. J., Haardt, M., et al. (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6, 482–497.PubMedCrossRefGoogle Scholar
  49. 49.
    Pollard, H., Toumaniantz, G., Amos, J. L., et al. (2001) Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids. J. Gene Med. 3, 153–164.PubMedCrossRefGoogle Scholar
  50. 50.
    Berntzen, G., Lunde, E., Flobakk, M., Andersen, J. T., Lauvrak, V., and Sandlie, I. (2005) Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells. J. Immunol. Methods 298, 93–104.PubMedCrossRefGoogle Scholar
  51. 51.
    Parham, J. H., Kost, T., and Hutchins, J. T. (2001) Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines. Cytotechnology 35, 181–187.CrossRefGoogle Scholar
  52. 52.
    McMahan, C. J., Slack, J. L., Mosley, B., et al. (1991) A novel IL-1 receptor, cloned from B cells by mammalian expression, is expresed in many cell types. EMBO J. 10, 2821–2832.PubMedGoogle Scholar
  53. 53.
    Giri, J. G., Ahdieh, M., Eisenman, J., et al. (1994) Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13, 2822–2830.PubMedGoogle Scholar
  54. 54.
    Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–74.PubMedCrossRefGoogle Scholar
  55. 55.
    Gorman, C. M., Gies, D., McCray, G., and Huang, M. (1989) The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology 171, 377–385.PubMedCrossRefGoogle Scholar
  56. 56.
    Cachianes, G., Ho, C., Weber, R. F., Williams, S. R., Goeddel, D. V., and Leung, D. W. (1993) Epstein-Barr virus-derived vectors for transient and stable expression of recombinant proteins. Biotechniques 15, 255–259.PubMedGoogle Scholar
  57. 57.
    DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., and Calos, M. P. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell Biol. 7, 379–387.PubMedGoogle Scholar
  58. 58.
    Lebkowski, J. S., Clancy, S., and Calos, M. P. (1985) Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression. Nature 317, 169–171.PubMedCrossRefGoogle Scholar
  59. 59.
    Lewis, E. D. and Manley, J. L. (1985) Repression of simian virus 40 early transcription by viral DNA replication in human 293 cells. Nature 317, 172–175.PubMedCrossRefGoogle Scholar
  60. 60.
    Cho, M. S., Yee, H., and Chan, S. (2002) Establishment of a human somatic hybrid cell line for recombinant protein production. J. Biomed. Sci. 9, 631–638.PubMedCrossRefGoogle Scholar
  61. 61.
    Cho, M. S., Yee, H., Brown, C., Mei, B., Mirenda, C., and Chan, S. (2003) Versatile expression system for rapid and stable production of recombinant proteins. Biotechnol. Prog. 19, 229–232.PubMedCrossRefGoogle Scholar
  62. 62.
    Kunaparaju, R., Liao, M., and Sunstrom, N. A. (2005) Epi-CHO, an episomal expression system for recombinant protein production in CHO cells. Biotechnol. Bioeng. 91, 670–677.PubMedCrossRefGoogle Scholar
  63. 63.
    Liao, M. and Sunstrom, N. A. (2006) A transient expression vector for recombinant protein production in Chinese hamster ovary cells. J. Chem. Tech. Biotech. 81, 82–88.CrossRefGoogle Scholar
  64. 64.
    Rosser, M. P., Xia, W., Hartsell, S., et al. (2005) Transient transfection of CHO-K 1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr. Purif. 40, 237–243.PubMedCrossRefGoogle Scholar
  65. 65.
    Tait, A. S., Brown, C. J., Galbraith, D. J., et al. (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol. Bioeng. 88, 707–721.PubMedCrossRefGoogle Scholar
  66. 66.
    Xia, W., Bringmann, P., McClary, J., et al. (2006) High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr. Purif. 45, 115–124.PubMedCrossRefGoogle Scholar
  67. 67.
    Cockett, M. I., Bebbington, C. R., and yarranton, G. T. (1991) The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucleic Acids Res. 19, 319–325.PubMedCrossRefGoogle Scholar
  68. 68.
    Mizuguchi, H., Hosono, T., and Hayakawa, T. (2000) Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells. FEBS Lett. 472, 173–178.PubMedCrossRefGoogle Scholar
  69. 69.
    Heffernan, M. and Dennis, J. W. (1991) Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells. Nucleic Acids Res. 19, 85–92.PubMedCrossRefGoogle Scholar
  70. 70.
    Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B., and Schaffner, W. (1985) A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521–530.PubMedCrossRefGoogle Scholar
  71. 71.
    Foecking, M. K. and Hofstetter, H. (1986) Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45, 101–105.PubMedCrossRefGoogle Scholar
  72. 72.
    Kim, D. W., Uetsuki, T., Kaziro, Y., Yamaguchi, N., and Sugano, S. (1990) Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Genet 91, 217–223.Google Scholar
  73. 73.
    Kim, S. Y., Lee, J. H., Shin, H. S., Kang, H. J., and Kim, Y. S. (2002) The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter. J. Biotechnol. 93, 183–187.PubMedCrossRefGoogle Scholar
  74. 74.
    McNeall, J., Sanchez, A., Gray, P. P., Chesterman, C. N., and Sleigh, M. J. (1989) Hyperinducible gene expression from a metallothionein promoter containing additional metal-responsive elements. Gene 76, 81–88.PubMedCrossRefGoogle Scholar
  75. 75.
    Huang, M. T. and Gorman, C. M. (1990) Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18, 937–947.PubMedCrossRefGoogle Scholar
  76. 76.
    Dolph, P. J., Huang, J. T., and Schneider, R. J. (1990) Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex. J. Virol. 64, 2669–2677.PubMedGoogle Scholar
  77. 77.
    Huang, W. and Flint, S. J. (1998) The tripartite leader sequence of subgroup C adenovirus major late mRNAs can increase the efficiency of mRNA export. J. Virol. 72, 225–235.PubMedGoogle Scholar
  78. 78.
    Kaufman, R. J. (1985) Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors. Proc. Natl. Acad. Sci. USA 82, 689–693.PubMedCrossRefGoogle Scholar
  79. 79.
    Logan, J. and Shenk, T. (1984) Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc. Natl. Acad. Sci. USA 81, 3655–3659.PubMedCrossRefGoogle Scholar
  80. 80.
    Svensson, C. and Akusjarvi, G. (1985) Adenovirus VA RNAI mediates a translational stimulation which is not restricted to the viral mRNAs. EMBO J. 4, 957–964.PubMedGoogle Scholar
  81. 81.
    Sclimenti, C. R. and Calos, M. P. (1998) Epstein-Barr virus vectors for gene expression and transfer. Curr. Opin. Biotechnol. 9, 476–479.PubMedCrossRefGoogle Scholar
  82. 82.
    Mackey, D. and Sugden, B. (1999) The linking regions of EBNA1 are essential for its support of replication and transcription. Mol. Cell Biol. 19, 3349–3359.PubMedGoogle Scholar
  83. 83.
    Tomiyasu, K., Satoh, E., Oda, Y., Nishizaki, K., Kondo, M., Imanishi, J., and Mazda, O. (1998) Gene transfer in vitro and in vivo with Epstein-Barr virus-based episomal vector results in markedly high transient expression in rodent cells. Biochem. Biophys. Res. Commun. 253, 733–738.PubMedCrossRefGoogle Scholar
  84. 84.
    Ceccarelli, D. F. and Frappier, L. (2000) Functional analyses of the EBNA1 origin DNA binding protein of Epstein-Barr virus. J. Virol. 74, 4939–4948.PubMedCrossRefGoogle Scholar
  85. 85.
    Gahn, T. A. and Sugden, B. (1995) An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene. J. Virol. 69, 2633–2636.PubMedGoogle Scholar
  86. 86.
    Reisman, D. and Sugden, B. (1986) Trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. Mol. Cell Biol. 6, 3838–3846.PubMedGoogle Scholar
  87. 87.
    Sugden, B. and Warren, N. (1988) Plasmid origin of replication of Epstein-Barr virus, oriP, does not limit replication in cis. Mol. Biol. Med. 5, 85–94.PubMedGoogle Scholar
  88. 88.
    Wu, H., Kapoor, P., and Frappier, L. (2002) Separation of the DNA replication, segregation, and transcriptional activation functions of Epstein-Barr nuclear antigen 1. J. Virol. 76, 2480–2490.PubMedCrossRefGoogle Scholar
  89. 89.
    Aiyar, A., Tyree, C., and Sugden, B. (1998) The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element. EMBO J. 17, 6394–6403.PubMedCrossRefGoogle Scholar
  90. 90.
    Calos, M. P. (1998) Stability without a centromere. Proc. Natl. Acad. Sci. USA 95, 4084–4085.PubMedCrossRefGoogle Scholar
  91. 91.
    Langle-Rouault, F., Patzel, V., Benavente, A., et al. (1998) Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids. J. Virol. 72, 6181–6185.PubMedGoogle Scholar
  92. 92.
    Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523.PubMedCrossRefGoogle Scholar
  93. 93.
    Stadler, J., Lemmens, R., and Nyhammar, T. (2004) Plasmid DNA purification. J. Gene Med. 6 Suppl 1, S54-S66.PubMedCrossRefGoogle Scholar
  94. 94.
    Wright, J., Jordan, M., and Wurm, F. (2001) Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection. Cytotechnology 35, 165–173.CrossRefGoogle Scholar
  95. 95.
    Schmid, G., Schlaeger, E. J., and Wipf, B. (2001) Non-GMP plasmid production for transient transfection in bioreactors. Cytotechnology 35, 157–164.CrossRefGoogle Scholar
  96. 96.
    Chu, G. and Sharp, P. A. (1981) SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen. Gene 13, 197–202.PubMedCrossRefGoogle Scholar
  97. 97.
    Song, W. and Lahiri, D. K. (1995) Efficient transfection of DNA by mixing cells in suspension with calcium phosphate. Nucleic Acids Res. 23, 3609–3611.PubMedCrossRefGoogle Scholar
  98. 98.
    Girard, P., Porte, L., Berta, T., Jordan, M., and Wurm, F. (2001) Calcium phosphate transfection optimization for serum-free suspension culture. Cytotechnology 35, 175–180.CrossRefGoogle Scholar
  99. 99.
    Lindell, J., Girard, P., Muller, N., Jordan, M., and Wurm, F. (2004) Calfection: a novel gene transfer method for suspension cells. Biochim. Biophys. Acta 1676, 155–161.PubMedGoogle Scholar
  100. 100.
    von Harpe, A., Petersen, H., Li, Y., and Kissel, T. (2000) Characterization of commercially available and synthesized polyethylenimines for gene delivery. J. Control Release 69, 309–322.CrossRefGoogle Scholar
  101. 101.
    Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Control Release 60, 149–160.PubMedCrossRefGoogle Scholar
  102. 102.
    Boussif, O., Zanta, M. A., and Behr, J. P. (1996) Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 3, 1074–1080.PubMedGoogle Scholar
  103. 103.
    Durocher, Y., Perret, S., and Kamen, A. (2001) Recombinant protein production by transient transfection of suspension-growing cells. In: Recombinant Protein Production With Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology. (Merten, O. W., Mattanovich, D., Lang, C., et al., eds.), Kluwer, Dordrecht, The Netherlands, pp. 329–335.Google Scholar
  104. 104.
    Wightman, L., Kircheis, R., Rossler, V., et al. (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J. Gene Med. 3, 362–372.PubMedCrossRefGoogle Scholar
  105. 105.
    Itaka, K., Harada, A., Yamasaki, Y., Nakamura, K., Kawaguchi, H., and Kataoka, K. (2004) In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J. Gene Med. 6, 76–84.PubMedCrossRefGoogle Scholar
  106. 106.
    Brunner, S., Furtbauer, E., Sauer, T., Kursa, M., and Wagner, E. (2002) Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol. Ther. 5, 80–86.PubMedCrossRefGoogle Scholar
  107. 107.
    Geisse, S., Jordan, M., and Wurm, F. M. (2005) Large-scale transient expression of therapeutic proteins in mammalian cells. Methods Mol. Biol. 308, 87–98.PubMedGoogle Scholar
  108. 108.
    Dee, K. U., Shuler, M. L., and Wood, A. (1997) Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production. Biotechnol. Bioeng. 54, 191–205.CrossRefPubMedGoogle Scholar
  109. 109.
    Jalkanen, M. (1987) Biology of cell surface heparan sulfate proteoglycans. Med. Biol. 65, 41–47.PubMedGoogle Scholar
  110. 110.
    Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) Regulated shedding of syndecan-1 and-4 ectodomains by thrombin and growth factor receptor activation. J. Biol. Chem. 272, 14,713–14,720.CrossRefGoogle Scholar
  111. 111.
    Legendre, J. Y., Trzeciak, A., Bohrmann, B., Deuschle, U., Kitas, E., and Supersaxo, A. (1997) Dioleoylmelittin as a novel serum-insensitive reagent for efficient transfection of mammalian cells. Bioconjug. Chem. 8, 57–63.PubMedCrossRefGoogle Scholar
  112. 112.
    Shi, C., Shin, Y. O., Hanson, J., Cass, B., Loewen, M. C., and Durocher, Y. (2005) Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells. Biochemistry 44, 15,705–15,714.Google Scholar
  113. 113.
    Farrell, P. and Iatrou, K. (2004) Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium. Protein Expr. Purif. 36, 177–185.PubMedCrossRefGoogle Scholar
  114. 114.
    Loomis, K. H., Yaeger, K. W., Batenjany, M. M., et al. (2005) InsectDirect System: rapid, high-level protein expression and purification from insect cells. J. Struct. Funct. Genomics 6, 189–194.PubMedCrossRefGoogle Scholar
  115. 115.
    Tonini, T., Claudio, P. P., Giordano, A., and Romano, G. (2004) Transient production of retroviral- and lentiviral-based vectors for the transduction of Mammalian cells. Methods Mol. Biol. 285, 141–148.PubMedGoogle Scholar
  116. 116.
    Zufferey, R. (2002) Production of lentiviral vectors. Curr. Top. Microbiol. Immunol. 261, 107–121.PubMedGoogle Scholar
  117. 117.
    Grimm, D. (2002) Production methods for gene transfer vectors based on adeno-associated virus serotypes. Methods 28, 146–157.PubMedCrossRefGoogle Scholar
  118. 118.
    Merten, O. W., Geny-Fiamma, C., and Douar, A. M. (2005) Current issues in adeno-associated viral vector production. Gene Ther. 12, S51-S61.PubMedCrossRefGoogle Scholar
  119. 119.
    Merten, O. W. (2004) State-of-the-art of the production of retroviral vectors. J. Gene Med. 6, S105-S124.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  1. 1.Laboratoire de Biotechnologie Vétérinaire et Alimentaire, Faculté de Médecine VétérinaireUniversité de MontréalQuébecSainte-HyacintheCanada
  2. 2.Biotechnology Research InstituteNational Research Council CanadaMontrealCanada

Personalised recommendations