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Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells.

ACS synthetic biology (2020-05-30)
Tao Sun, Wee Chiew Kwok, Koon Jiew Chua, Tat-Ming Lo, Jason Potter, Wen Shan Yew, Jonathan D Chesnut, In Young Hwang, Matthew Wook Chang
ABSTRACT

Chinese hamster ovary (CHO) cells are the superior host cell culture models used for the bioproduction of therapeutic proteins. One of the prerequisites for bioproduction using CHO cell lines is the need to generate stable CHO cell lines with optimal expression output. Antibiotic selection is commonly employed to isolate and select CHO cell lines with stable expression, despite its potential negative impact on cellular metabolism and expression level. Herein, we present a novel proline-based selection system for the isolation of stable CHO cell lines. The system exploits a dysfunctional proline metabolism pathway in CHO cells by using a pyrroline-5-carboxylate synthase gene as a selection marker, enabling selection to be made using proline-free media. The selection system was demonstrated by expressing green fluorescent protein (GFP) and a monoclonal antibody. When GFP was expressed, more than 90% of stable transfectants were enriched within 2 weeks of the selection period. When a monoclonal antibody was expressed, we achieved comparable titers (3.35 ± 0.47 μg/mL) with G418 and Zeocin-based selections (1.65 ± 0.46 and 2.25 ± 0.07 μg/mL, respectively). We further developed a proline-based coselection by using S. cerevisiae PRO1 and PRO2 genes as markers, which enables the generation of 99.5% double-transgenic cells. The proline-based selection expands available selection tools and provides an alternative to antibiotic-based selections in CHO cell line development.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-ALDH18A1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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IgG from human serum, reagent grade, ≥95% (SDS-PAGE), essentially salt-free, lyophilized powder
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Anti-Human IgG (Fab specific)−Peroxidase antibody produced in goat, affinity isolated antibody