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

Biotechnology and Bioengineering
Fabiana FernandesPaula M Alves

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 app...Continue Reading

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Citations

Dec 18, 2013·Journal of Biotechnology·Fabiana FernandesPaula M Alves
Jul 28, 2015·Biotechnology Journal·Ana F RodriguesAna S Coroadinha
Oct 29, 2013·Journal of Biotechnology·João VidigalAna P Teixeira
Aug 27, 2013·Insect Biochemistry and Molecular Biology·Dingpei LongZhonghuai Xiang
Dec 21, 2017·Scientific Reports·Quang Vinh PhanManfred Gossen
Nov 17, 2017·Applied Microbiology and Biotechnology·João VidigalAna P Teixeira
Feb 19, 2013·Expert Review of Vaccines·Fabiana FernandesPaula M Alves
Aug 19, 2020·Nanomaterials·Eduard Puente-MassaguerFrancesc Gòdia
Oct 15, 2018·Applied Microbiology and Biotechnology·Eduard Puente-MassaguerFrancesc Gòdia
Nov 29, 2020·Biotechnology Journal·Eduard Puente-MassaguerFrancesc Gòdia
Aug 23, 2020·Insect Biochemistry and Molecular Biology·Xien ChenSubba Reddy Palli
Nov 8, 2019·Journal of Biotechnology·Bárbara FernandesAntónio Roldão
May 14, 2021·G3 : Genes - Genomes - Genetics·Mafalda M DiasAntónio Roldão
Jul 17, 2020·Journal of Biotechnology·Eduard Puente-MassaguerMartí Lecina
Mar 20, 2019·Industrial & Engineering Chemistry Research·Christine M YeeFei Wen

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