RCME: Mammalian genome engineering

A significant challenge for producing any protein in mammalian production cell lines (i.e. CHO) with stable high-level performance consists in the difficulty to insert recombinant genes in a well-producing spot in the host genome. Only a small number of cell lines are commercially available for targeted genomic insertion. An extensive selection of mammalian cell lines, ideally tailored to the specific requirements of a particular protein to be produced, is largely lacking to date. This is partly due to a lack of tools for rapidly generating cell lines containing defined and well-producing spots in their genome for integrating one or more genes of interest.

The Helmholtz Centre for Infection Research (HZI) has developed a superior technology for generating stably modified master cell lines which contain an integration site at a validated, well-producing hot-spot which is engineered to accept foreign genes of interest into a locus occupied by a fluorescent protein marker in the master cell line. High-level production master cell lines are isolated by preparative sorting of cells expressing green fluorescent protein from the site of integration on the genome. Different master cell lines are selected for high, medium and low level expression of the fluorescent marker, and thoroughly validated by means of monitoring fluorescent protein expression over generations. Then, the green fluorescent protein gene can be rapidly exchanged for a heterologous gene of interest by using recombinase-mediated cassette exchange (RMCE). In this way, novel production cell lines can be quickly and easily obtained from the resulting master cells [Wilke et al., PLOS One 2011]. Genome engineering by RMCE thus allows inserting transgenes of interest precisely into a defined expression hot-spot of the host cell genome.

Timeline for exchanging the Gene of Interest (GOI) by RMCE.

Following transfection, selection and subcloning,  stable cells are analyzed by flow cytometry (FC) andPCR to confirm the cassette exchange. Recombinant protein production can be detected using Western blots (WB) after 6 weeks. It takes 7 weeks from transfection of the master cells to cryopreservation of the first aliquot of cell clones with integrated Gene of Interest (GOI).