The aim of this proposal is to produce a cost effective kit that will facilitate the production of mouse embryonic stem (ES) cell/chimeras to produce genetically engineered mice. The kit will be designed for use by investigators with only limited experience with mouse embryo culture and embryo transfer. Costs will be reduced by eliminating the need for local animal colonies for the production of 8-cell mouse embryos by supplying them from a central stock of frozen embryos. The thawed 8-cell embryos will be aggregated with the ES cells and then transferred into the uteri of pseudopregnant surrogate mothers. Improvement of the embryo culture systems used in the several steps of the procedure will be made by optimizing the culture medium KSOM AA/Gluc, a new very effective culture medium for preimplantation mouse embryos. The feasibility study in this Phase I application will test the following hypotheses: (1) KSOM AA/Gluc can replace Dulbecco's modification of Eagle's minimum essential medium for the culture of embryonic stem cells, (2) Thawed, previously frozen, 8-cell embryos, cultured in KSOM AA/Gluc develop into blastocysts that can then develop into normal fetuses after transfer to the uterus of surrogate mothers, and (3) Aggregates of embryonic stem cells with thawed, previously frozen, 8-cell embryos, cultured in KSOM AA/Gluc, can develop into newborn mice that are embryonic stem cell/blastocyst derived chimeras.
