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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-6011-1) contains supplementary material, which is available to authorized users.
Highlights
Monoclonal antibodies represent the main market fraction of all biotherapeutics with USD 24.6 billion in US sales and a growth of 18.2 % in 2012 (Aggarwal 2014)
The use of two heterospecific, noncompatible FLP recognition target sites in combination with a screening/selection marker enables the isolation of an engineered host cell line containing a chromosomal recombinant cassette in a predefined, transcriptionally active locus
Our contribution describes the generation of a new host cell line capable to integrate different transgenes into the same chromosomal locus driven by the same transcriptional control elements
Summary
Monoclonal antibodies (mAb) represent the main market fraction of all biotherapeutics with USD 24.6 billion in US sales and a growth of 18.2 % in 2012 (Aggarwal 2014). Together with residual vector-specific components, this effect is a frequent cause of different epigenetic silencing events, among these are histone deacetylation, distinct histone methylation or phosphorylation steps, and DNA-/promoter-methylation patterns (Mutskov and Felsenfeld 2004; Richards and Elgin 2002). These effects may be triggered by integration of the transgene into heterochromatin, leading to the loss or reduction of expression (Kwaks and Otte 2006). The established DUKX-B11 F3/F cell line provides a stable, retargetable chromosomal locus, indicated by homogenous intracellular green fluorescent protein (gfp) expression, capable to exchange different transgenes into the same transcription control region by site-specific recombination. ScFv-Fc clones developed by RMCE showed reproducible transcription and secretion levels demonstrating the suitability of DUKX-B11 F3/F for comparison of different recombinant producer cell lines
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call