In vivo siRNA delivery to the mouse hypothalamus confirms distinct roles of TR beta isoforms in regulating TRH transcription

Abstract

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for evaluating gene function in vivo. In particular it should be able to provide tissue-specific and developmental stage-specific knock-down of target genes in physiological contexts. However, demonstrations of its use on neuronal specific genes in vivo are lacking. We examined whether a recently developed cationic lipid based approach was applicable to study the differential effects of the two beta thyroid hormone receptor (TR) isoforms, TRβ1 and TRβ2, on T 3-transcriptional repression of the hypothalamic gene, TRH. The cationic lipid based technique used, JetSI™/DOPE, was previously shown to efficiently knock-down reporter gene mRNA in vivo. Here we now show that its use to vectorise siRNA against TRβ1 and TRβ2 mRNA abrogates T 3-mediated repression of hypothalamic TRH transcription. In particular, when using siRNA against either TRβ1 or TRβ2 differential effects are revealed. siRNA directed against TRβ1 blocks both T 3 independent activation and T 3 dependent modulation of TRH transcription. In contrast, siRNA directed against TRβ2 abrogates only T 3 repression of transcription. These results corroborate our previous findings obtained in mutant TRβ −/− mice, showing that the TRβ1 and TRβ2 isoforms have differential effects on T 3- TRH transcription. The data thus show that the cationic lipid-based siRNA strategy can effectively be used to reveal fine, tissue specific and isoform specific effects on neuronal gene transcription in vivo.