Development and recent progress in the pronuclear injection-based targeted transgenesis method by site-specific recombination in mice

摘要

Pronuclear injection (PI) is the most common method for generating transgenic (Tg) mice, despite large variability in the level and pattern of transgene expression because of its dependence on the gene copy number, configuration and integration site. In this study, we established a Pi-based targeted transgenesis (PITT) system based on site-specific recombination in fertilized eggs but not in embryonic stem (ES) cells. Seed mice were initially generated through ES cell targeting to contain loxP-site derivatives at a predetermined locus. A transgene, flanked by two mutant loxP sites, was then integrated into target loci in the seed mice by Cre-loxP recombination in fertilized eggs. Using this method, we have established more than 25 Tg lines, including fluorescent gene transgenic mice that exhibit ubiquitous, strong and reproducible transgene expression. The efficiency of correct integration per newborn using the PITT method was 4%-5% for Cre plasmid injection and 20% for Cre mRNA injection when 10-14-kb donor vectors were used, and there was a high germline transmission rate (>90%). We also demonstrated that knockdown mice can be readily generated by PITT by taking advantage of our reproducible and highly efficient expression system. Thus, the PITT method provides a strong basis for systematically generating a variety of targeted Tg lines for reliable transgene expression and gene knockdown, without using ES cells.