Citation

  • Authors: Guissouma, H., Froidevaux, M. S., Hassani, Z., Demeneix, B. A.
  • Year: 2006
  • Journal: Neurosci Lett 406 240-3
  • Applications: in vivo / DNA, siRNA / jetSI 10 mM

Method

jetSI and DOPE were mixed to DNA complexes at a 1.8 nitrogen/phosphate ratio (0.18 µl 10 mM jetSI and 0.18 µl DOPE 20 mM/µg of plasmid DNA, the resulting jetSI and DOPE ratio being 1:2). The injection mix was prepared for 20 mice as follows. Add 9 µl of jetSI/DOPE, 10 µl glucose 25 % to 50 µl (tube 1). Add 5 µl of DNA (5 µg/µl), 5 µl siRNA (20 µM), 10 µl glucose 25 % to 50 µl (tube 2). Add tube 1 to tube 2, vortex and inject 4 µl per new born mouse into the hypothalamus. By using co-transfection experiments in vivo, they showed that siRNA directed against TRbeta1 and TRbeta2 abrogates T3 mediated repression on hypothalamic 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, TRbeta1 and TRbeta2, 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 TRbeta1 and TRbeta2 mRNA abrogates T3-mediated repression of hypothalamic TRH transcription. In particular, when using siRNA against either TRbeta1 or TRbeta2 differential effects are revealed. siRNA directed against TRbeta1 blocks both T3 independent activation and T3 dependent modulation of TRH transcription. In contrast, siRNA directed against TRbeta2 abrogates only T3 repression of transcription. These results corroborate our previous findings obtained in mutant TRbeta(-/-) mice, showing that the TRbeta1 and TRbeta2 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.

Pubmed