Generation of chimeras by morula aggregation.

This chapter describes the tools and the experimental route of targeted manipulation by aggregation in the mouse using targeted embryonic stem cells (ES cells). Instead of injecting ES cells into the blastocoel of a diploid blastocyst-stage embryo (3.5 dpc) ES cells can be brought together with diploid morula-stage embryos (2.5 dpc). The zona pellucida of the embryo needs to be removed and one or two embryos (sandwich aggregation) are put together with ES cells into an indentation well of a cell culture grade dish overnight for aggregation. This can be performed manually using a stereomicroscope and does not require any special training or expensive instrumentation.The next day, the embryo would have developed into a blastocyst in vitro and can be transferred to a pseudopregnant female mouse (see Chapter 15 ).The use of tetraploid embryos generated by electrofusion will lead to entirely ES cell-derived fetuses.