Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Abstract

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies.

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

“…It was expected that this combination would work well under an excision-free condition, as previously observed by araki et al and thomson et al [3,69]. As expected, the in vitro analysis confirmed that this combination of loxP sites was completely exclusive [47]. We further generated "seed mice" with this set of loxP sites at two genomic loci, H2-Tw3 and Rosa26.…”

“…2A). In our study, we used loxP sequences JT15/JTZ17 (inverted-repeat variants) and lox2272 (spacer variant) and placed them in the same orientation [46,47] (Fig. 2A).…”

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

“…We did not observe any obvious change in HB9-GFP transgene expression upon removal of the bacterial backbone; this observation could be the consequence of small numbers of animals compared, or a result of the possibility that the bacterial backbone could have different effects on different promoters. The effect of bacterial backbone has been reported for randomly integrated and episomal transgenes (34)(35)(36)(37), and other native bacterial sequences like the lacZ gene or the neomycin resistance gene have been linked to variegation in transgenic animals (40,(42)(43)(44). Our experiments based on site-specific integration enabled us to systematically and quantitatively characterize these effects for single-copy chromosomally integrated transgenes.…”

“…Recently, two other approaches for site-specific transgenesis in mice using pronuclear injection were reported (39)(40)(41). One approach relied on zinc-finger nucleases to create site-specific double stranded breaks that were repaired by injected recombinant DNA via homologous recombination (39,41).…”

“…Both reports have yet to demonstrate germline transmission and proper adult expression of the transgenes, although proper germline transmission for the same technique in rats was reported (41). The other approach used the Cre recombinase to catalyze cassette exchange in the Rosa26 locus and a tissue-specific locus, H2-Tw3, at an average frequency of approximately 4.3%, with proper expression and germline transmission of the transgenes (40). One drawback of the Cre-based method is that it cannot be used to generate Cre-activated transgenes (containing loxP-STOP-loxP), which are frequently used for reporting Cre activity and perturbing gene function in cells in which Cre is expressed.…”