Study of the Endogenous CRAC Channel Using shRNA-Mediated Gene Silencing.

The Ca2+ release-activated Ca2+ (CRAC) current is a major signaling event in non-excitable cells whereby Ca2+ store depletion activates Ca2+ entry across the plasma membrane from the extracellular space. Stromal interaction molecule 1 (STIM1) and Orai1 proteins are the key molecular players of the CRAC channel. Previous studies have linked activity of this channel to many physiological functions, and dysregulation of the CRAC channel has been associated with various diseases. In the absence of inducible tissue-specific knockout mice, in vivo knockdown studies examining the endogenous function of CRAC channel proteins, STIM1 and Orai1, are a challenge. In this chapter, we describe a lentiviral delivery system of shRNA-mediated gene silencing that has proven successful in studying the endogenous CRAC channel in vivo.