3 years ago

Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange

Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange
Kevin Kayser, Scott Bahr, Natalie Sealover, Elisabeth Gludovacz, Nan Lin, Martina Baumann, Henry George, Nicole Borth
Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site. Recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-defined genomic locations. Baumann et al. describe a strategy making use of alternate start codons for the surface reporter protein CD4 to sort for CHO cells with highest transcription rates in combination with RMCE for the subsequent exchange of CD4 against a GOI. Deep sequencing of the final cell pool identified a single genome integration site, despite the absence of any subcloning step.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26388

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