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

Abstract：

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Vijaya Chandra SH,Makhija H,Peter S,Myint Wai CM,Li J,Zhu J,Ren Z,D'Alcontres MS,Siau JW,Chee S,Ghadessy FJ,Dröge P

doi

10.1093/nar/gkv1345

subject

Has Abstract

pub_date

2016-04-07 00:00:00

pages

e55

issue

6

eissn

0305-1048

issn

1362-4962

pii

gkv1345

journal_volume

44

pub_type

杂志文章

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