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Expression of a Secreted Protein in Mammalian Cells Using Baculovirus Particles

  • Barbara Ann Jardin
  • Cynthia B. EliasEmail author
  • Satya Prakash
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

Key words

CMV promoter BacMam Baculovirus Full factorial design Design of experiment Hollow fiber Tangential flow filtration 

References

  1. 1.
    Boyce FM, Bucher NLR. Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 1996;93:2348–52.Google Scholar
  2. 2.
    Lehtolainen P, Tyynela K, Kannasto J, et al. Baculoviruses exhibit restricted cell type specificity in rat brain: a comparison of baculovirus- and adenovirus-mediated intracerebral gene transfer in vivo. Gene Therapy 2002;9:1693–9.Google Scholar
  3. 3.
    Grassi G, Kohn H, Dapas B, et al. Comparison between recombinant baculo- and adenoviral-vectors as transfer system in cardiovascular cells. Archives of Virology 2006;151:255–71.Google Scholar
  4. 4.
    Martyn JC, Dong X, Holmes-Brown S, et al. Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus. Archives of Virology 2007;152:329–43.Google Scholar
  5. 5.
    Salminen M, Airenne KJ, Rinnankoski R, et al. Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes. Journal of Virology 2005;79:2720–8.Google Scholar
  6. 6.
    Bilello JP, Cable EE, Myers RL, Isom HC. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatopytes. Gene Therapy 2003;10:733–49.Google Scholar
  7. 7.
    Barsoum J, Brown R, Mckee M, Boyce FM. Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Human Gene Therapy 1997;8:2011–8.Google Scholar
  8. 8.
    Sandig V, Hofmann C, Steinert S, et al. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Human Gene Therapy 1996;7:1937–45.Google Scholar
  9. 9.
    Nicholson LJ, Philippe M, Paine AJ, et al. RNA interference mediated in human primary cells via recombinant baculoviral vectors. Molecular Therapy 2005;11:638–44.Google Scholar
  10. 10.
    Lee HP, Ho YC, Hwang SM, et al. Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression. Biotechnology and Bioengineering 2007;97:649–55.Google Scholar
  11. 11.
    Ho YC, Chung YC, Hwang SM, et al. Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells. Journal of Gene Medicine 2005;7:860–8.Google Scholar
  12. 12.
    Airenne KJ, Hiltunen MO, Turunen MP, et al. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Therapy 2000;7:1499–504.Google Scholar
  13. 13.
    Ma L, Tamarina N, Wang Y, et al. Baculovirus-mediated gene transfer into pancreatic islet cells. Diabetes 2000;49:1986–91.Google Scholar
  14. 14.
    Kinnunen K, Kalesnykas G, Mähönen AJ, et al. Baculovirus is an efficient vector for the transduction of the eye: comparison of baculovirus- and adenovirus-mediated intravitreal vascular endothelial growth factor D gene transfer in the rabbit eye. J Gene Med 2009.Google Scholar
  15. 15.
    Spenger A, Ernst W, Condreay JP, et al. Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells. Protein Expression and Purification 2004;38:17–23.Google Scholar
  16. 16.
    Gheshlaghi R, Scharer JM, Moo-Young M, Douglas PL. Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods. Biotechnology and Bioengineering 2005;90: 754–60.Google Scholar
  17. 17.
    Berger J, Hauber J, Hauber R, et al. Secreted Placental Alkaline-Phosphatase - A Powerful New Quantitative Indicator of Gene-Expression in Eukaryotic Cells. Gene 1988;66:1–10.Google Scholar
  18. 18.
    Condreay JP, Witherspoon SM, Clay WC, Kost TA. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proceedings of the National Academy of Sciences of the United States of America 1999;96:127–32.Google Scholar
  19. 19.
    Jardin BA, Montes J, Lanthler S, et al. High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf-9-BEV system. Biotechnology and Bioengineering 2007;97 :332–45.Google Scholar
  20. 20.
    Bedard C, Tom R, Kamen A. Growth, Nutrient Consumption, and End-Product Accumu-lation in Sf-9 and Bti-Eaa Insect-Cell Cultures - Insights Into Growth Limitation and Metabolism. Biotechnology Progress 1993;9: 615–24.Google Scholar
  21. 21.
    Elias CB, Carpentier E, Durocher Y, et al. Improving glucose and gluamiue metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme. Biotechnology Progress 2003;19:90–7.Google Scholar
  22. 22.
    Zeiser A, Bedard C, Voyer R, et al. On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements. Biotechnology and Bioengi-neering 1999;63:122–6.Google Scholar
  23. 23.
    Zeiser A, Elias CB, Voyer R, et al. On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process. Biotechnology Progress 2000;16:803–8.Google Scholar
  24. 24.
    Jardin BA, Zhao Y, Selvaraj M, et al. Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system. Journal of Biotechnology 2008;135:272–80.Google Scholar
  25. 25.
    Janakiraman V, Forrest WF, Chow B, Seshagiri S. A rapid method for estimation of baculovirus titer based on viable cell size. Journal of Virological Methods 2006;132:48–58.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Barbara Ann Jardin
    • 1
    • 2
  • Cynthia B. Elias
    • 3
    Email author
  • Satya Prakash
    • 1
    • 2
  1. 1.Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Artificial Cells and Organs Research CenterMcGill UniversityMontrealCanada
  2. 2.Biomedical Technology and Cell Therapy Research Laboratory, Department of Physiology, Artificial Cells and Organs Research CenterMcGill UniversityMontrealCanada
  3. 3.Bulk Manufacturing/Cells and Viral Media, Sanofi-PasteurTorontoCanada

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