Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology

  • João Vidigal
  • Fabiana Fernandes
  • Ana S. Coroadinha
  • Ana P. Teixeira
  • Paula M. Alves
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1104)

Abstract

Traditional cell line development is quite laborious and time-consuming as it is based on the random integration of the gene of interest which leads to unpredictable expression behavior. In opposition, recombinase-mediated cassette exchange systems represent a powerful genetic engineering approach, allowing site-specific insertion of recombinant genes into pre-tagged genomic loci with superior expression characteristics, thus bypassing the need for extensive clone screening and shortening the development timelines. Such systems have not been widely implemented in insect cell lines used for the production of recombinant proteins most commonly through the baculovirus expression vector system. Herein, it is provided the protocol for the implementation of a FLP-mediated cassette exchange system in Spodoptera frugiperda Sf 9 cells, in order to grant a flexible cell line for the stable production of recombinant proteins.

Key words

Insect cell line development Flipase-mediated cassette exchange Stable expression of recombinant proteins 

References

  1. 1.
    Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575CrossRefGoogle Scholar
  2. 2.
    McCarroll L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotechnol 8:590–594CrossRefGoogle Scholar
  3. 3.
    van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107:3–15CrossRefGoogle Scholar
  4. 4.
    Jarvis DL, Fleming JA, Kovacs GR et al (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Biotechnology (N Y) 8:950–955CrossRefGoogle Scholar
  5. 5.
    Van Oers MM, Thomas AA, Moormann RJ et al (2001) Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells. J Biotechnol 86:31–38CrossRefGoogle Scholar
  6. 6.
    Harrison RL, Jarvis DL (2007) Transforming lepidopteran insect cells for continuous recombinant protein expression. Methods Mol Biol 388:299–2316Google Scholar
  7. 7.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398CrossRefGoogle Scholar
  8. 8.
    Kromenaker SJ, Srienc F (1994) Stability of producer hybridoma cell lines after cell sorting: a case study. Biotechnol Prog 10:299–307CrossRefGoogle Scholar
  9. 9.
    Sternberg N, Sauer B, Hoess R et al (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. J Mol Biol 187:197–212CrossRefGoogle Scholar
  10. 10.
    Buchholz F, Angrand PO, Stewart AF (1996) A simple assay to determine the functionality of cre or FLP recombination targets in genomic manipulation constructs. Nucleic Acids Res 24:3118–3119CrossRefGoogle Scholar
  11. 11.
    Schaft J, Ashery-Padan R, van der Hoeven F et al (2001) Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31:6–10CrossRefGoogle Scholar
  12. 12.
    Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95:5505–5510CrossRefGoogle Scholar
  13. 13.
    Schlake T, Bode J (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746–12751CrossRefGoogle Scholar
  14. 14.
    Qiao J, Oumard A, Wegloehner W et al (2009) Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. J Mol Biol 390:579–594CrossRefGoogle Scholar
  15. 15.
    Turan S, Galla M, Ernst E et al (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 407:193–221CrossRefGoogle Scholar
  16. 16.
    Coroadinha AS, Schucht R, Gama-Norton L et al (2006) The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange. J Biotechnol 124:457–468CrossRefGoogle Scholar
  17. 17.
    Fernandes F, Vidigal J, Dias MM et al (2012) Flipase-mediated cassette exchange in Sf 9 insect cells for stable gene expression. Biotechnol Bioeng 109:2836–2844CrossRefGoogle Scholar
  18. 18.
    Baer A, Bode J (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr Opin Biotechnol 12:473–480CrossRefGoogle Scholar
  19. 19.
    Sorrell DA, Robinson CJ, Smith JA et al (2010) Recombinase mediated cassette exchange into genomic targets using an adenovirus vector. Nucleic Acids Res 38:e123CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • João Vidigal
    • 1
    • 2
  • Fabiana Fernandes
    • 1
    • 2
  • Ana S. Coroadinha
    • 1
    • 2
  • Ana P. Teixeira
    • 1
    • 2
  • Paula M. Alves
    • 1
    • 2
  1. 1.Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  2. 2.iBETInstituto de Biologia Experimental e TecnológicaOeirasPortugal

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