Chimeric Mouse Generation by ES Cell Blastocyst Microinjection and Uterine Transfer.

The ability to generate chimeric mice through microinjecting embryonic stem (ES) cells into blastocysts is a critical step for the conventional ES cell-mediated knockout technology. In recent years, designer nuclease-based methods, especially the CRISPR technology, have substantially decreased the needs for blastocyst microinjection. However, this method has still remained as a valuable technique for generating sophisticated genetic models as well as for stem cell research. In this chapter, we describe the detailed procedures used in our laboratory on how to use ES cells to produce chimeric mice, including derivation and inactivation of MEF feeder cells, culturing and handling of mouse ES cells, collection and microinjection of blastocysts, and finally implantation of injected blastocysts into the uteri of pseudopregnant surrogate mothers.