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