Please use this identifier to cite or link to this item: https://doi.org/10.1021/acssynbio.0c00221
Title: Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells
Authors: Sun, Tao 
Kwok, Wee Chiew 
Chua, Koon Jiew 
Lo, Tat-Ming 
Potter, Jason
Yew, Wen Shan 
Chesnut, Jonathan D
Hwang, In Young 
Chang, Matthew Wook 
Keywords: Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
CHO cells
coselection
monoclonal antibody
proline metabolism
P5CS
RECOMBINANT PROTEIN-PRODUCTION
EXPRESSION
LINES
GENOME
SENSITIVITY
Issue Date: 2020
Publisher: AMER CHEMICAL SOC
Citation: Sun, Tao, Kwok, Wee Chiew, Chua, Koon Jiew, Lo, Tat-Ming, Potter, Jason, Yew, Wen Shan, Chesnut, Jonathan D, Hwang, In Young, Chang, Matthew Wook (2020). Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells. ACS SYNTHETIC BIOLOGY 9 (7) : 1864-1872. ScholarBank@NUS Repository. https://doi.org/10.1021/acssynbio.0c00221
Abstract: © 2020 American Chemical Society. 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.
Source Title: ACS SYNTHETIC BIOLOGY
URI: https://scholarbank.nus.edu.sg/handle/10635/177211
ISSN: 21615063
21615063
DOI: 10.1021/acssynbio.0c00221
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