Gene Silencing in Mouse Embryonic Stem Cells
Embryonic stem cells (ESCs) are promising tools for regenerative medicine as well as for biotechnological research. However, to exploit ESCs for clinical purposes, a better understanding of the molecular mechanisms that control the pluripotency and differentiation of ESCs is required. Several extrinsic signaling pathways contribute to the maintenance of pluripotency, as well as induction of differentiation, in ESCs. However, the mechanisms that regulate extrinsic signaling in ESCs are largely unknown. Heparan sulfate (HS) is present ubiquitously as a component of cell surface proteoglycans and is known to play crucial roles in the regulation of several signaling pathways. We have validated that RNA interference (RNAi) is a useful method for the functional analysis of some target genes in mouse ESCs (mESCs). Indeed, we have investigated the functions of HS in mESCs by using RNAi and have demonstrated that HS on mESCs is involved in regulating signaling pathways that are important for the maintenance of mESCs. In this chapter, we describe detailed methods for the gene silencing of proteoglycan-related genes in mESCs by RNAi.
Key wordsRNA interference Short hairpin RNA Mouse embryonic stem cells EXT1 Heparan sulfate
We thank Prof. Toshio Kitamura and Prof. Kumiko Ui-Tei for gifting experimental materials. Our research was partially supported by funds from the MEXT, a Grant-in-Aid for Scientific Research (B) to SN, 20370051, 2008–2010, and from MEXT, the Matching Fund for Private Universities, S0901015, 2009–2014.