Establishing Mammalian Production Cell Lines for Structural Biology by Site-Specific Recombination

Abstract

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 Lec3.2.8.1 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.