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J Neurosci Methods. 2008 May 15;170(1):56-66. doi: 10.1016/j.jneumeth.2007.12.018. Epub 2008 Jan 6.

Development of an in vivo adeno-associated virus-mediated siRNA approach to knockdown tyrosine hydroxylase in the lateral retrochiasmatic area of the ovine brain.

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  • 1UMR85 Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France. dufourny@tours.inra.fr

Abstract

We developed a new technique of gene knockdown (KD) in a specific brain area of the ewe using an adeno-associated virus (AAV)-mediated short interfering RNA (siRNA) method to elucidate the importance of key factors of seasonal reproduction. Two 19-nucleotide sequences (TH1 or TH2) were chosen from the tyrosine hydroxylase (TH) gene. TH1, TH2 or a random sequence (TH3) was incorporated into an eGFP expressing AAV vector. Firstly, 5 microl of AAV-TH1 or AAV-TH2 solutions (8-9 x 10(11)Vg/ml) were stereotaxically injected into one A15 nucleus while the other received a control treatment. Ewes were killed after 15 or 75 days. The number of TH neurons was 49% and 36% lower on the AAV-TH1 treated side than on the control side 15 and 75 days post-injection, respectively. AAV-TH2 did not induce a significant variation in TH cell population. Finally, in order to increase the KD, two groups of ewes received 10 microl of AAV-TH1 either in a bolus injection or in two 5 microl inoculations carried out 2 weeks apart. Only ewes receiving a bolus injection showed a larger KD reaching 66% 2 months after inoculation. This method proved effective in reducing TH expression and will be further developed to understand cellular mechanisms driving seasonal functions.

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