Targeted transgenesis through pronuclear injection of improved vectors into in vitro fertilized eggs

We recently described a new transgenic technique termed pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010), which enables targeted integration of transgenes into predetermined loci (e.g. Rosa26) by Cre-loxP-based recombination in fertilized eggs, leading to stable and reproducible transgene expression. In this study, two steps of PITT were modified. The donor vector was improved by introducing an FLPe expression cassette (Fig. 1a). Therefore, selfremoval of the extra sequence flanked by FRT sequences occurs without additional FLPe administration. In addition, the plasmid backbone of the donor vector was modified from pUC119-to pBR322based to reduce plasmid instability, which is sometimes associated with genomic DNA fragment cloning into a high-copy plasmid. Moreover, in vitro fertilized eggs were used in this study; in the previous study, fertilized eggs obtained by natural mating were used. In this study, fertilized eggs ([100) were obtained simultaneously using epididymal spermatozoa isolated from a single male carrying the targeted allele. Results are listed in Fig. 1b. Three independent donor vectors, pAWK, pAWV and pAXV, were microinjected with a Cre expression vector into in vitro fertilized eggs. The resulting PITT efficiency was 5/93 (5.4%; 95% CI, 1.8–12.1%), which is similar to that reported previously (4.3%; 95% CI, 2.7–6.5%), indicating the feasibility of this modified method and reproducibility of PITT. Regarding removal of the extra sequence, the recombinasemediated cassette exchange (RMCE) allele was successfully created in 60% of founder mice (3/5; indicated by *). Although the other two founder mice did not possess the RMCE allele, the extra sequences of these founders can be successfully removed by crossing them with an FLPe deleter mouse (Ohtsuka et al. 2010). Notably, all tested lines (5/5) exhibited successful germline transmission Electronic supplementary material The online version of this article (doi:10.1007/s11248-011-9505-y) contains supplementary material, which is available to authorized users.