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ACS Synth Biol. 2018 Sep 21;7(9):2148-2159. doi: 10.1021/acssynbio.8b00140. Epub 2018 Aug 16.

Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation.

Author information

1
The Novo Nordisk Foundation Center for Biosustainability , Technical University of Denmark , 2800 Kgs. Lyngby , Denmark.
2
Department of Molecular Science and Technology , Ajou University , Suwon 16499 , Republic of Korea.
3
Department of Pediatrics , University of California, San Diego , La Jolla , California 92093 , United States.
4
The Novo Nordisk Foundation Center for Biosustatainability , University of California, San Diego , La Jolla , California 92093 , United States.
5
Department of Biotechnology and Biomedicine , Technical University of Denmark , 2800 Kgs. Lyngby , Denmark.
6
Australian Institute for Bioengineering and Nanotechnology , University of Queensland , St Lucia , QLD 4072 , Australia.
7
Department of Biological Sciences , KAIST , Daejeon 34141 , Republic of Korea.

Abstract

Mammalian cells are widely used to express genes for basic biology studies and biopharmaceuticals. Current methods for generation of engineered cell lines introduce high genomic and phenotypic diversity, which hamper studies of gene functions and discovery of novel cellular mechanisms. Here, we minimized clonal variation by integrating a landing pad for recombinase-mediated cassette exchange site-specifically into the genome of CHO cells using CRISPR and generated subclones expressing four different recombinant proteins. The subclones showed low clonal variation with high consistency in growth, transgene transcript levels and global transcriptional response to recombinant protein expression, enabling improved studies of the impact of transgenes on the host transcriptome. Little variation over time in subclone phenotypes and transcriptomes was observed when controlling environmental culture conditions. The platform enables robust comparative studies of genome engineered CHO cell lines and can be applied to other mammalian cells for diverse biological, biomedical and biotechnological applications.

KEYWORDS:

CRISPR/Cas9; clonal variation; mammalian cells; recombinase-mediated cassette exchange; targeted integration; transcriptome

PMID:
30060646
DOI:
10.1021/acssynbio.8b00140

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