Lentiviral delivery of RNAi in hippocampal neurons.

The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 2003; Elbashir et al., 2001; Hannon et al., 2004). Effective delivery of siRNA molecules into target cells or tissues is critical for successful RNA interference (RNAi) application. Here, we describe the use of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors for delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to suppress gene expression. Major advantages of lentiviral vectors are their ability to transduce nondividing cells and to confer long-term expression of transgenes. This chapter covers selection of short hairpin sequences, vector design, production of lentiviral supernatants, transduction of dissociated primary hippocampal neurons, and testing the effectiveness of shRNA-mediated silencing.