dc.contributor.authorMakhija, Harshyaa
dc.contributor.authorRoy, Suki
dc.contributor.authorHoon, Shawn
dc.contributor.authorGhadessy, Farid John
dc.contributor.authorWong, Desmond
dc.contributor.authorJaiswal, Rahul
dc.contributor.authorCampana, Dario
dc.contributor.authorDröge, Peter
dc.identifier.citationMakhija, 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/gky500en_US
dc.description.abstractAdvances 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.en_US
dc.description.sponsorshipASTAR (Agency for Sci., Tech. and Research, S’pore)en_US
dc.description.sponsorshipNMRC (Natl Medical Research Council, S’pore)en_US
dc.format.extent14 p.en_US
dc.relation.ispartofseriesNucleic Acids Researchen_US
dc.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 (http://creativecommons.org/licenses/by-nc/4.0/), 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 journals.permissions@oup.comen_US
dc.subjectProtein Expressionen_US
dc.subjectDRNTU::Science::Biological sciencesen_US
dc.titleA novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expressionen_US
dc.typeJournal Article
dc.contributor.researchNTU Institute of Structural Biologyen_US
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.description.versionPublished versionen_US

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