Gene silencing using RNA interference in embryonic stem cells.
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.
Velkey
J Matthew
JM
Department of Cell and Development Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
Slawny
Nicole A
NA
Gratsch
Theresa E
TE
O'Shea
K Sue
KS
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology*; D005456:Fluorescent Dyes; D005821:Genetic Techniques; D007150:Immunohistochemistry; D008055:Lipids; D008081:Liposomes; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D005456:Fluorescent Dyes; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D035683:MicroRNAs; D034741:RNA, Small Interfering