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Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy. [ABSTRACT FROM AUTHOR]

Differentiation of pluripotent embryonic stem (ES) cells into specific lineages is an important source of cells for implantation and gene delivery, as well as a useful model to study patterns of differentiation and gene expression during the very early development of the mammalian embryo (1). Embryonic stem cells are derived from the blastocyst inner cell mass (2,3), and remain totipotent when grown on the surface of embryonic fibroblasts or on gelatincoated substrates in the presence of leukemia inhibitory factor (LIF). ES cells appear to have unlimited proliferative capability, and, remarkably, when returned to the inner cell mass after culture and gene manipulation, resume their development and participate fully in the formation of ALL tissue types. Recently, embryonic stem cells have been derived from human blastocysts after in vitro fertilization (IVF) (4,5). Pluripotent stem cells have also been derived from human primordial germ cells (6), with obvious clinical applications. [ABSTRACT FROM AUTHOR]