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Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange
Author(s) -
Baumann Martina,
Gludovacz Elisabeth,
Sealover Natalie,
Bahr Scott,
George Henry,
Lin Nan,
Kayser Kevin,
Borth Nicole
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26388
Subject(s) - chinese hamster ovary cell , subcloning , biology , reporter gene , start codon , gene , codon usage bias , genome , transcription (linguistics) , genetics , microbiology and biotechnology , plasmid , gene expression , cell culture , messenger rna , linguistics , philosophy
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.