Development of an in vivo adeno-associated virus-mediated siRNA approach to knockdown tyrosine hydroxylase in the lateral retrochiasmatic area of the ovine brain.
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.
Dufourny
Laurence
L
UMR85 Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France. dufourny@tours.inra.fr
Migaud
Martine
M
Thiery
Jean-Claude
JC
Malpaux
Benoit
B
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000033:anatomy & histology / Q000201:enzymology*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D009897:Optic Chiasm / Q000033:anatomy & histology / Q000201:enzymology*; D010052:Ovariectomy; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012098:Reproduction / Q000502:physiology; D012621:Seasons; D012756:Sheep; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase