Skip to main content

Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology

  • Protocol
  • First Online:
Animal Cell Biotechnology

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575

    Article  CAS  Google Scholar 

  2. McCarroll L, King LA (1997) Stable insect cell cultures for recombinant protein production. Curr Opin Biotechnol 8:590–594

    Article  Google Scholar 

  3. van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107:3–15

    Article  Google Scholar 

  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) 8:950–955

    Article  CAS  Google Scholar 

  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 86:31–38

    Article  Google Scholar 

  6. Harrison RL, Jarvis DL (2007) Transforming lepidopteran insect cells for continuous recombinant protein expression. Methods Mol Biol 388:299–2316

    CAS  Google Scholar 

  7. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398

    Article  CAS  Google Scholar 

  8. Kromenaker SJ, Srienc F (1994) Stability of producer hybridoma cell lines after cell sorting: a case study. Biotechnol Prog 10:299–307

    Article  CAS  Google Scholar 

  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 187:197–212

    Article  CAS  Google Scholar 

  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 24:3118–3119

    Article  CAS  Google Scholar 

  11. Schaft J, Ashery-Padan R, van der Hoeven F et al (2001) Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31:6–10

    Article  CAS  Google Scholar 

  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 95:5505–5510

    Article  CAS  Google Scholar 

  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 33:12746–12751

    Article  CAS  Google Scholar 

  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 390:579–594

    Article  CAS  Google Scholar 

  15. Turan S, Galla M, Ernst E et al (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 407:193–221

    Article  CAS  Google Scholar 

  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 124:457–468

    Article  CAS  Google Scholar 

  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 109:2836–2844

    Article  CAS  Google Scholar 

  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 12:473–480

    Article  CAS  Google Scholar 

  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:e123

    Article  Google Scholar 

Download references

Acknowledgments

We thank Mafalda M. Dias (ITQB-UNL/IBET) for help with clone stability analysis. This work was supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT) through the Projects PTDC/EBB-EBI/102266/2008 and PTDC/EBB-EBI/102750/2008 and by the European Commission (FP7/2007-2013, grant agreement N° 270089). João Vidigal and Fabiana Fernandes also thank FCT for their PhD fellowships (SFRH/BD/90564/2012 and SFRH/BD/43830/2008, respectively).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Vidigal, J., Fernandes, F., Coroadinha, A.S., Teixeira, A.P., Alves, P.M. (2014). Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Molecular Biology, vol 1104. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-733-4_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-733-4_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-732-7

  • Online ISBN: 978-1-62703-733-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics