title: Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells.
abstract: Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.
authors.affiliation: Department of Biotechnology/Vienna Institute of BioTechnology (BOKU-VIBT), University of Natural Resources and Life Sciences (Vienna), Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: Patrick
authors.initials: P
authors.lastname: Mayrhofer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kratzer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sommeregger
authors.forename: Willibald
authors.initials: W
authors.lastname: Steinfellner
authors.forename: David
authors.initials: D
authors.lastname: Reinhart
authors.forename: Alexander
authors.initials: A
authors.lastname: Mader
authors.forename: Soeren
authors.initials: S
authors.lastname: Turan
authors.forename: Junhua
authors.initials: J
authors.lastname: Qiao
authors.forename: Juergen
authors.initials: J
authors.lastname: Bode
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D020869:Gene Expression Profiling; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies