Chinese hamster ovary (CHO) cell lines for biopharmaceutical production are usually established by random transgene integration, resulting in a heterogenous cell population with various transgene copy number and integration sites. In addition, cell growth, protein productivity, and quality may be affected by the transgene insertion sites in the genome. Therefore, identifying the genomic insertion site of transgenes will help better characterization of cell lines. While next-generation sequencing is routinely employed for identifying the insertion site, it requires complicated processing and analysis of sequence reads. Moreover, it is a time-consuming (4-6 weeks) and costly ($2,000-3,000 per project) technique, demanding simple, easy, and cheap methods for gene integration site analysis. There are PCR-based techniques for isolating and amplifying genomic DNA sequences neighboring the DNA junction of integrants such as Alu-PCR, A-T linker PCR and splinkerette-PCR. We explored the feasibility of these PCR-based techniques in CHO genome to identify the integration site of transgene with less resources (both time and cost).  As a proof-of-concept study, we digested a plasmid vector using restriction enzymes and ligated them with splinkerette adaptors that prevent adaptor-adaptor amplification. Next, we conducted PCR and identified a band with an expected size, and verified the amplified DNA using restriction enzyme mapping. This technique was then applied to CHO genomic DNA samples to identify the integration sites of a recombinant protein gene. In conclusion, PCR-based assays would help to provide the genomic information of CHO production cells with more affordable options in the process of cell line development.<br/>

PCR-based assays for identifying transgene insertion sites in CHO genome

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.