Abstract

The stimulation of inositol phospholipid metabolism via phospholipase C (PLC) is an important signal transduction pathway in a wide variety of cell types. Activation of the pathway is associated with many aspects of cellular activity, including cell growth and differentiation. Activation of hormone-sensitive PLC results in the rapid breakdown of polyphosphoinositides to generate two second messengers: inositol trisphosphate and diacylglycerol. The water-soluble inositol trisphosphate is involved in the release of intracellular calcium from internal stores, whereas the lipophilic diacylglycerol is involved in protein kinase C activation. Inositol supplementation is essential for the in vitro growth of rabbit blastocysts, and studies have shown that the components of the signaling system are present in mouse and cattle embryos and in mouse embryonic stem (ES) cells. In ES cells, the signaling system appears to be constitutively active and essential for normal ES cell proliferation. Here, we describe in detail the materials required and some of techniques involved in studying the phosphoinositide signaling system in mouse ES cells. Furthermore, we describe methods of analyzing the effects of modulating the PtdIns signaling system on ES cell proliferation and the induction of apoptosis.