Inducible H1 Promoter-Driven Lentiviral siRNA Expression by Stuffer Reporter Deletion

Abstract

Introduction of 19–23-bp small interfering RNA (siRNA) into mammalian cells has become a standard procedure to downregulate mRNA with high efficacy. siRNAs can be introduced into cells either as synthetic duplexes or as hairpin structures produced by Pol III promoter-driven vectors. Pol III promoter-expressed small hairpin RNAs (shRNAs) offer a great possibility for the production of endogenous siRNA, which can be used for stable siRNA production in vivo. A major drawback of this strategy is the incapability of detecting rapidly occurring cellular responses. Here, we present a lentiviral shRNA-producing vector system, which can be induced by CRE recombinase enzyme to overcome these limitations. Following the addition of CRE, the pLIND (LentiINDucible) will activate siRNA production by deleting EGFP and a stop cassette between the promoter and siRNA oligo. Target gene downregulation capacity was comparable to that of a noninducible siRNA system.