Abstract
Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre–loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.
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Acknowledgement
We thank H. Miura, S. Ogiwara, and T. Sato (Tokai Univ.) for technical assistance. We are grateful to M. Sato (Kagoshima Univ.) for critical reading of the manuscript. This work was supported by the Grant-in-Aid for Young Scientists (B) (20700368) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; by Takeda Science Foundation; by the A-STEP (Adaptable and Seamless Technology Transfer Program through Target-driven R&D) from Japan Science and Technology Agency (JST); and by 2011-2012 Tokai University School of Medicine, Project Research to M.O.
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Ohtsuka, M. (2014). Development of Pronuclear Injection-Based Targeted Transgenesis in Mice Through Cre–loxP Site-Specific Recombination. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1215-5_1
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DOI: https://doi.org/10.1007/978-1-4939-1215-5_1
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