Derivation and manipulation of trophoblast stem cells from mouse blastocysts

The trophoblast is the first lineage to undergo differentiation during mammalian development. In the preimplantation blastocyst embryo, two cell types are present including the inner cell mass (ICM) and the trophectoderm (TE). ICM cells exhibit pluripotent potential, or the capacity to give rise to all cells represented in the adult organism, while TE cells are multipotent and are therefore only capable of differentiating into trophoblast lineages represented in the placenta. The TE is essential for implantation of the embryo into the uterine tissue, formation of trophoblast lineages represented in the placenta, and exchange of nutrients and waste between the embryo and the mother. Trophoblast stem (TS) cells, which can be derived from the TE of preimplantation embryos in the presence of external signals such as FGF4, can self-renew indefinitely, and because they are capable of differentiating into epithelial lineages of the trophoblast, TS cells are a useful in vitro model to study the biology of the trophoblast including epigenetic regulation of gene expression. In this chapter we describe protocols for derivation of TS cells from mouse blastocysts, culture conditions that promote self-renewal and differentiation, and methods to transduce TS cells with lentiviral particles encoding shRNAs. These protocols are sufficient for efficient derivation of TS cells and robust RNAi knockdown of target genes in TS cells.