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Gewählte Publikation:

Baumann, M; Gludovacz, E; Sealover, N; Bahr, S; George, H; Lin, N; Kayser, K; Borth, N.
(2017): Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange
BIOTECHNOL BIOENG. 2017; 114(11): 2616-2627. FullText FullText_BOKU

Abstract:
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.
Autor/innen der BOKU Wien:
Baumann Martina
Borth Nicole
BOKU Gendermonitor:


Find related publications in this database (Keywords)
alternative start codons
Chinese hamster ovary (CHO) cells
genomic integration site
recombinase mediated cassette exchange (RMCE)


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