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. 2016 Feb 15;95:3-12.
doi: 10.1016/j.ymeth.2015.11.022. Epub 2015 Dec 2.

A Method for Specifically Targeting Two Independent Genomic Integration Sites for Co-Expression of Genes in CHO Cells

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A Method for Specifically Targeting Two Independent Genomic Integration Sites for Co-Expression of Genes in CHO Cells

Bahar Baser et al. Methods. .

Abstract

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.

Keywords: Binary RMCE expression system; CHO; CHO Lec3.2.8.1; Chinese hamster ovary cells; FRT; Flp; Multi-RMCE; Recombinase mediated cassette exchange.

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