SBIR-STTR Award

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.

Thesaurus Terms:
biomedical equipment development, embryo /fetus culture growth media embryo /fetus preservation, embryo /fetus transplantation, laboratory mouse

The aim of this proposal is to produce a cost-effective kit that will facilitate the production of mouse embryonic stem (ES) cell / blastocyst chimeras to make genetically engineered mice. The kit will be designed so that it can be used by investigators with only limited experience with mouse embryo culture and embryo transfer. Costs of making knock-out mice will be significantly 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 to the uteri of pseudopregnant surrogate mothers. In Phase II of this application, the following research will be done to produce an optimized kit: (1) a method will be developed to assay rapidly the aggregation of ES cells with 8-cell embryos; (2) KSOM/AA, a new medium that is very effective for the culture of mouse preimplantation embryos, will be optimized for the aggregation of ES cells and mouse embryos; (3) the culture of ES cells on a feeder layer will be improved by replacing the feeder layer with a plate of supporting killed primary mouse embryonic fibroblasts; (4) KSOM/AA will be modified to replace Dulbecco's MEM for the culture of ES cells; and (5) data will be collected to determine which of two inbred strains of mouse (C57BL/6 or FVB/N) are preferable as donors of the 8-cell embryos used for aggregation.

Thesaurus Terms:
biomedical equipment development, early embryonic stage, embryo /fetus culture, embryonic stem cell, tissue mosaicism growth media embryo /fetus preservation, embryo /fetus transplantation, laboratory mouse