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
Date Issued
April 2018
Journal
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Research Laboratory of Electronics
Massachusetts Institute of Technology. Synthetic Biology Center
Terms of Use
Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1093/nar/gky216
