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The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.

Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.