A multi-landing pad DNA integration platform for mammalian cell engineering
Author(s)
Wroblewska, Liliana; Nelson, Tom; Zhang, Xin; Liu, Yan; Lowe, Alexis; Bandara, Kalpanie; Baijuraj, Swetha; Zhang, Lin; Gaydukov, Leonid A.; Teague, Brian Paul; Jagtap, Kalpana P; Mamo, Selamawit W; Tseng, Wen Allen; Das, Jishnu; Summers, Nevin M; Lu, Timothy K; Weiss, Ron; ... Show more Show less
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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.
Date issued
2018-04Department
Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Research Laboratory of Electronics; Massachusetts Institute of Technology. Synthetic Biology CenterJournal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Gaidukov, Leonid et al. “A Multi-Landing Pad DNA Integration Platform for Mammalian Cell Engineering.” Nucleic Acids Research 46, 8 (April 2018): 4072–4086 © 2018 The Author(s)
Version: Final published version
ISSN
0305-1048
1362-4962