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Title: A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression
Authors: Makhija, Harshyaa
Roy, Suki
Hoon, Shawn
Ghadessy, Farid John
Wong, Desmond
Jaiswal, Rahul
Campana, Dario
Dröge, Peter
Keywords: Transgenesis
Protein Expression
DRNTU::Science::Biological sciences
Issue Date: 2018
Source: Makhija, H., Roy, S., Hoon, S., Ghadessy, F. J., Wong, D., Jaiswal, R., . . . Dröge, P. (2018). A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression. Nucleic Acids Research, 46(16), e99-. doi:10.1093/nar/gky500
Series/Report no.: Nucleic Acids Research
Abstract: Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.
ISSN: 0305-1048
DOI: 10.1093/nar/gky500
Schools: School of Biological Sciences 
Research Centres: NTU Institute of Structural Biology 
Rights: © 2018 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

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