Article

Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression

  1. Fabiana Fernandes1,2,
  2. João Vidigal1,2,
  3. Mafalda M. Dias2,
  4. Kristala L.J. Prather3,
  5. Ana S. Coroadinha1,2,
  6. Ana P. Teixeira1,2,*,
  7. Paula M. Alves1,2

Article first published online: 19 MAY 2012

DOI: 10.1002/bit.24542

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 109, Issue 11, pages 2836–2844, November 2012

Additional Information

How to Cite

Fernandes, F., Vidigal, J., Dias, M. M., Prather, K. L.J., Coroadinha, A. S., Teixeira, A. P. and Alves, P. M. (2012), Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression. Biotechnol. Bioeng., 109: 2836–2844. doi: 10.1002/bit.24542

Author Information

  1. 1

    Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; telephone: +351 214469417; fax: +351 214421161

  2. 2

    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República 2780-157 Oeiras, Portugal

  3. 3

    Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

*Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; telephone: +351 214469417; fax: +351 214421161.

Publication History

  1. Issue published online: 24 SEP 2012
  2. Article first published online: 19 MAY 2012
  3. Accepted manuscript online: 7 MAY 2012 07:21AM EST
  4. Manuscript Accepted: 24 APR 2012
  5. Manuscript Revised: 21 MAR 2012
  6. Manuscript Received: 8 FEB 2012

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Keywords:

  • transformed Sf9 cell line;
  • RMCE;
  • recombinant proteins;
  • flipase

Abstract

Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research. Biotechnol. Bioeng. 2012; 109: 2836–2844. © 2012 Wiley Periodicals, Inc.

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