Article

Site-specific integrase-mediated transgenesis in mice via pronuclear injection.

The Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2011; 108(19):7902-7. DOI: 10.1073/pnas.1019507108
Source: PubMed

ABSTRACT Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

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