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 o f the most important techniques for production of knockout or transgenic mi ce. However, skillful manipulation techniques and tremendous effort are req uired for this method. To overcome this difficulty, we applied a piezo-micr omanipulator (PMM), which has been used for intracytoplasmic sperm injectio n 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, 9 1% of the blastocysts were manipulated successfully. Using the PMM signific antly (P < 0.01) increased the success rate of ES injection to 97%. The num ber of embryos manipulated in an hour increased from 9.7 embryos with the c onventional method to 27.0 embryos with the PMM method. The injected ES cel ls 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 ch imeric mice with the conventional method, whereas 42% of the neonates were chimeric after the PMM method. These results indicate that microinjection o f the ES cells into blastocysts is more efficient by the PMM method than th e conventional method.