Using TARGATT™ Technology to Generate Site-Specific Transgenic Mice
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The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as “TARGATT™” (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.
Key wordsPronuclear microinjection PhiC31 integrase TARGATT™ Site-specific transgenic Rosa26 knockin H11 locus attP attB
The author would like to thank all Applied StemCell, Inc. employees who have contributed to improving the TARGATT™ technologies. Thanks also go to our customers who entrusted us on generating TARGATT™ models for their research.
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