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.Why Would I Want Embryonic Stem Cells?
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.
ES lines are generated from individually cultured preimplantation embryos (essentially as described in Bryja et al., 2006 An efficient method for derivation of mouse embryonic stem cells. Stem Cells 24:844). Blastocyst stage embryos are placed a layer of feeder fibroblasts where they attach and grow out. Individual outgrowths are manually disaggregated, plated on fresh feeder fibroblasts, grown and embryonic stem cell colonies are identified by their morphology. ES colonies are picked, passed and expanded to prepare frozen stocks and genomic DNA.What Factors Contribute to Success?
You should plan to have enough practiced males (10) and enough young, reproductively mature females (25) to produce sufficient embryos for success. For example, C57BL/6J mice have an average litter size of 6. Only 30% of embryos are expected to give rise to ES cell lines. In a heterozygote intercross, only one quarter of embryos are expected to be homozygous mutant. Thus, you should provide us with a minimum of 5 females plugged on the same day (5 females x 6 embryos/female x .33 lines/embryo x .25 homozygotes/embryo = ~2.5 homozygous mutant ES cell lines). Since the estrus cycle is 4-5 days, a stock of 25 females and 10 males is recommended to provide 5 females plugged on the same day. If you are skilled at identifying females in estrus fewer males could be used, but the same number of females would be required.
It will be best for your analysis of different cell lines if the genetic background is uniform, and the only known genetic difference is the genotype at the gene of interest. ES cell lines can be made from most inbred strains of mice. The 129 strain of mice is best at producing ES cells which are competent to contribute to germ cells, but germ line transmission is not the goal for most ES lines made from mutants. We have been successful in making ES cell lines from the 129, C57BL/6J and C3H/He inbred strains, and others have made lines from CBA, DBA, BALB/c and FVB.
Very robust and genetically identical ES cell lines can be made from the embryos of a cross between two different inbred strains (an F1 cell line), but requires that you have your mutation(s) on two different backgrounds (e.g. 129 and C57BL/6) if you want homozygous mutant lines.