Long double-stranded RNA-mediated RNA interference as a tool to achieve site-specific silencing of hypothalamic neuropeptides

Abstract

RNA interference (RNAi) has become a popular tool to silence gene expression in a variety of in vitro and in vivo systems. However, it has met with limited success in inhibiting gene expression in adult mammals. Here we demonstrate that long double-stranded RNA (dsRNA) can be used to create a "site-specific", transient knockdown of genes in a fashion that is phenotypically akin to genetically manipulated organisms. Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) that regulate a variety of physiological processes including the hypothalamic-pituitary-adrenal axis (HPA axis), energy and water homeostasis were used as model systems. Stereotaxic injections of dsRNA against CRF and AVP in the PVN specifically abolished the expression of these genes in the PVN leaving expression in other loci intact. Control dsRNA did not affect CRF or AVP expression in any brain region, suggesting that dsRNA did not shut down global protein synthesis. ANOVA showed significant main effects of silencing of CRF on dampening of the stress-activated release of adrenocorticotrophin hormone (ACTH) (F(2,7)=4.87; p<0.047). Silencing of AVP resulted in increased water consumption, increased urine output and decreased urine osmolality as compared to control dsRNA-treated rats. Furthermore, dsRNA had no obvious deleterious effects on body weight or food consumption, variables considered essential in ruling out adverse physiologic effects in animal models. Thus, using long dsRNA, we were able to ascertain site-specific roles of CRF and AVP in adult rats without any developmental compensation and in a wild-type background.