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

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 T3-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 T3-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 T3 independent activation and T3 dependent modulation of TRH transcription. In contrast, siRNA directed against TRβ2 abrogates only T3 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 T3-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.