Specific knock-down of GAD67 in the striatum using naked small interfering RNAs.
Small interfering RNAs (siRNA) are double-stranded RNAs of 9-29 nucleotides designed to reduce the expression of homologous genes by a process known as RNA interference (RNAi). Most studies using siRNA in neurons have been performed in mammalian cell cultures. Only few reports have reported the effects of in vivo infusion of siRNA into the brain. In the present study, we performed local intracerebral infusions of naked siRNA against glutamic acid decarboxylase 67 (GAD67) mRNA to engineer specific knock-down of GAD67 protein in the striatum of adult rats. Directly injecting a mix of GAD67 siRNAs into the striatum decreased the levels of corresponding mRNA, evaluated by quantitative real-time PCR. In particular, we show that GAD67 mRNA expression is reduced in the striatum for 3, 6, and 24h following intrastriatal injection of GAD67 siRNA and is restored at 72h. Relative to controls, the levels of GAD67 protein were also lower in the striatum for 6 and 24h after injection. No changes in GAD65 expression, one of the two isoforms of GAD, were detected in the striatum, which further validates the specificity of the siRNA. We demonstrate the efficiency of the RNAi strategy for producing a specific and selective down-regulation of GAD67 in the adult rat brain. This suggests that siRNA-mediated gene knock-down constitute a valid methodological approach for studying the functional consequences of a transient decrease of a gene expression in a brain structure.
Manrique
Christine
C
Aix-Marseille Universités, CNRS UMR6149, Marseille 13331, France. christine.manrique@univ-provence.fr
Compan
Valérie
V
Rosselet
Céline
C
Duflo
Sylvie Gustave Dit
SG
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D051381:Rats; D020318:Rats, Long-Evans; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1