title: Site-specific integrase-mediated transgenesis in mice via pronuclear injection.
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.
authors.affiliation: The Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Bosiljka
authors.initials: B
authors.lastname: Tasic
authors.forename: Simon
authors.initials: S
authors.lastname: Hippenmeyer
authors.forename: Charlene
authors.initials: C
authors.lastname: Wang
authors.forename: Matthew
authors.initials: M
authors.lastname: Gamboa
authors.forename: Hui
authors.initials: H
authors.lastname: Zong
authors.forename: Yanru
authors.initials: Y
authors.lastname: Chen-Tsai
authors.forename: Liqun
authors.initials: L
authors.lastname: Luo
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008845:Microinjections; D011247:Pregnancy; D011994:Recombinant Proteins / Q000235:genetics; D014018:Tissue Distribution
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019426:Integrases