Conservative site-specific and single-copy transgenesis in human LINE-1 elements.

Autor:	Vijaya Chandra SH; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551., Makhija H; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551., Peter S; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551., Myint Wai CM; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551., Li J; Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China., Zhu J; Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China., Ren Z; Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Tonghe GuangZhou 510515, People's Republic of China State Key Laboratory of Organ Failure Research, Division of Nephrology, Nanfang Hospital, Tonghe, Guangzhou 510515, People's Republic of China., D'Alcontres MS; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551., Siau JW; p53Lab, Agency for Science Technology and Research, Singapore 138673., Chee S; p53Lab, Agency for Science Technology and Research, Singapore 138673., Ghadessy FJ; p53Lab, Agency for Science Technology and Research, Singapore 138673., Dröge P; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 pdroge@ntu.edu.sg.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2016 Apr 07; Vol. 44 (6), pp. e55. Date of Electronic Publication: 2015 Dec 15.
DOI:	10.1093/nar/gkv1345
Abstrakt:	Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, term edattH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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