Embryonic stem (ES) cell lines derive from the inner cell mass of the blastocyst stage embryo. The inner cell mass normally gives rise to the entire embryo and part of the placenta. ES cell lines, when combined with a host embryo to make a chimeric animal, contribute to the same cell types, including germ cells. In vitro, ES cells normally are grown on a layer of feeder fibroblasts which provide factors necessary for maintenance of the stem cell state. Embryonic stem cell lines can be made from mutant mice, and cellular phenotypes can be examined in detail in vitro. ES cells proliferate rapidly, form colonies, transfect well, grow clonally after transfection, and can be differentiated into a wide variety of cell types in vitro and in vivo. ES cells divide about once every 12 hours, and if they were plated as single cells, form clonal colonies. ES cells can be transfected with high efficiency by electroporation. Transient and stable transfections are easy to perform. shRNA, lentiviral vectors, and gene targeting work well in ES cells. ES cells can be differentiated into a large number of embryonic and extraembryonic cell types in vitro, including extraembryonic endoderm, vasculature, cardiac myocytes, blood cells, neurons, etc. Although many in vitro differentiation protocols give rise to mixed cell types, refined protocols for selection of specific cell types are developed frequently. ES cells can be grown as tumors in immunocompromised hosts or immunoprotected sites where they will differentiate into derivatives of all three germ layers. Both X chromosomes are active in female ES cells, and random X chromosome inactivation occurs upon differentiation.
The gene expression and chromatin profiles of undifferentiated and differentiated ES cells are well characterized, as are the genes and factors which are required for maintenance of the stem cell state.
The TMII will isolate blastocysts from uterine horns of up to 5 pregnant E5 females. Blastocysts (between 10-40) will be rinsed in DMEM and placed onto an inactivated SNL76 (STO) feeder cell layer in embryo growth media plus LIF. The blastocysts will be grown ex vivo in CO2 incubator for approximately 5 days, at which time the embryos will have hatched and the inner cell mass will have expanded. Clumps of inner cell mass cells will be disaggregated by trypsin, and individual clones of ES cells established in 96-well micro-titer dishes bearing inactivated SNL76 (STO) feeder cells. The Core will expand, select clones for freeze down and DNA preparation, and will provide the DNA to the Investigator for further analysis. 4. Frozen vials of the properly targeted clones will be transferred to the Investigator. 5. Although the Core will proceed with due care and diligence, no guarantee can be made regarding the viability or pluripotency of each embryo-derived ES cell line.
The success rate of this procedure is very high as long as blastocysts can be obtained from the mouse strain in question. The ideal age for of in-house blastocyst donors for superovulation response is 28 +/- 2 days