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A multi-landing pad DNA integration platform for mammalian cell engineering

Nucleic acids research, 2018-05, Vol.46 (8), p.4072-4086 [Peer Reviewed Journal]

The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. 2018 ;ISSN: 0305-1048 ;EISSN: 1362-4962 ;DOI: 10.1093/nar/gky216 ;PMID: 29617873

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Title:
A multi-landing pad DNA integration platform for mammalian cell engineering
Author: Gaidukov, Leonid ; Wroblewska, Liliana ; Teague, Brian ; Nelson, Tom ; Zhang, Xin ; Liu, Yan ; Jagtap, Kalpana ; Mamo, Selamawit ; Tseng, Wen Allen ; Lowe, Alexis ; Das, Jishnu ; Bandara, Kalpanie ; Baijuraj, Swetha ; Summers, Nevin M ; Lu, Timothy K ; Zhang, Lin ; Weiss, Ron
Subjects: Animals ; Cell Engineering ; CHO Cells ; Cricetulus ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Genetic Loci ; Genome ; Homologous Recombination ; Molecular Biology ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - genetics ; Transgenes
Is Part Of: Nucleic acids research, 2018-05, Vol.46 (8), p.4072-4086
Description: Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.
Publisher: England: Oxford University Press
Language: English
Identifier: ISSN: 0305-1048
EISSN: 1362-4962
DOI: 10.1093/nar/gky216
PMID: 29617873
Source: PubMed Central (Open access)
DOAJ Directory of Open Access Journals
Geneva Foundation Free Medical Journals at publisher websites
Oxford University Press Open Access
MEDLINE

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A multi-landing pad DNA integration platform for mammalian cell engineering

The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. 2018 ;ISSN: 0305-1048 ;EISSN: 1362-4962 ;DOI: 10.1093/nar/gky216 ;PMID: 29617873

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