Application of the piezo-micromanipulator for injection of embryonic stem cells into mouse blastocysts.

Microinjection of embryonic stem (ES) cells into mouse blastocysts is one of the most important techniques for production of knockout or transgenic mice. However, skillful manipulation techniques and tremendous effort are required for this method. To overcome this difficulty, we applied a piezo-micromanipulator (PMM), which has been used for intracytoplasmic sperm injection in mice and production of cloned mice, for the injection of ES cells into blastocysts. When ES cells were injected by using a conventional method, 91% of the blastocysts were manipulated successfully. Using the PMM significantly (P < 0.01) increased the success rate of ES injection to 97%. The number of embryos manipulated in an hour increased from 9.7 embryos with the conventional method to 27.0 embryos with the PMM method. The injected ES cells did not show any detrimental effects due to a pulse from the PMM. After embryo transfer of the manipulated blastocysts, 39% of the newborns were chimeric mice with the conventional method, whereas 42% of the neonates were chimeric after the PMM method. These results indicate that microinjection of the ES cells into blastocysts is more efficient by the PMM method than the conventional method.