Establishing Mammalian Production Cell Lines for Structural Biology by Site-Specific Recombination
Mammalian cell culture techniques are becoming more and more important for recombinant protein production in structural studies. In particular, crystallography requires large amounts of high-quality protein. Unfortunately, establishing stable mammalian producer cell lines is a slow and expensive process. Strategies involving fluorescence-activated cell sorting (FACS) and site-specific recombination promise improvement. In this study, different Flp recombinase-mediated strategies were applied on a glycosylation mutant CHO Lec18.104.22.168 cell line. Stable cell lines were generated with a GFP reporter gene and FACS selection of fluorescent cells. We routinely obtained cell lines with stable high-level GFP expression over several months. Depending on the strategy, we either exchanged GFP in the master cell line against another gene by recombinase-mediated cassette exchange (RMCE) or excised GFP by site-specific recombination, thereby putting the gene of interest (GOI) under control of the promoter. Establishing a production cell line from a master cell line by RMCE took about 1 to 2 months while the GFP excision method required 4 months. The combination of FACS and site-specific recombination enabled fast and reproducible cloning of protein producer cell lines for structural biology that are stable without antibiotics.
KeywordsExchange Vector Production Cell Line Master Cell Line Human Hepatocyte Growth Factor G418 Resistent Coloni
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