Abstract
We recently reported a novel transgenic (Tg) technique named pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010; Ohtsuka et al. 2012a). The PITT technique enables targeted integration of a single-copy transgene into a specified locus by Cre-loxP mediated recombination and it guarantees faithful and reproducible transgene expression. As described in the first report, the PITT technique requires co-injection of a Cre expression plasmid and a donor vector containing loxP sequences into pronuclei of zygotes obtained from the seed mice that carry loxP sequences at the Rosa26 or H2-Tw3 loci. This method has a (targeted) integration efficiency of 26/585 (4.4 %; 95 % CI 2.9–6.4 %) (Ohtsuka et al. 2012b). Although PITT has a lower transgenic rate (4.4 %) when compared to traditional transgenesis (*10–20 % (Fielder and Montoliu 2011)), it is superior to the latter method since just obtaining one transgenic founder is sufficient in case of PITT. With an aim to improve the PITT technique further we attempted to co-inject iCre mRNA [codon improved Cre recombinase; (Shimshek et al. 2002)] into zygotes with the expectation that the Cre protein would be available more readily in the zygotes from injected iCre mRNA compared to when the Creexpression plasmid DNA was co-injected. We coinjected 10 ng/ll of pAOM donor vector (Ohtsuka et al. 2010) along with various concentrations of iCre mRNA (1–45 ng/ll) into zygotes obtained from the Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9703-x) contains supplementary material, which is available to authorized users.
Published Version
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