Published Online
on December 27, 2005

A more recent version of this article appeared on February 1, 2006

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Submitted on August 23, 2005
Revised on September 29, 2005

Adenovirus-Mediated Small-Interference RNA for In Vivo Silencing of Angiotensin AT_1a Receptors in Mouse Brain

Yanfang Chen^*; Hao Chen; Andrea Hoffmann; David R. Cool; Debra I. Diz; Mark C. Chappell; Alex Chen; and Mariana Morris

From the Department of Pharmacology and Toxicology (Y.C., H.C., A.H., D.R.C., M.M.), Wright State University Boonshoft School of Medicine, Dayton, Ohio; Hypertension and Vascular Disease Center (D.I.D., M.C.C.), Wake Forest University School of Medicine, Winston Salem, NC; and Department of Pharmacology and Toxicology (A.C.), Michigan State University School of Medicine, East Lansing, Mich.

^* To whom correspondence should be addressed. E-mail: yanfang.chen{at}wright.edu.

Abstract--Because of the lack of pharmacological approaches, molecular genetic methods have been required to differentiate between angiotensin type 1(AT₁) receptor subtypes AT_1a and AT_1b. RNA interference is a new tool for the study of gene function, producing specific downregulation of protein expression. In this study, we used the small hairpin RNA (shRNA) cassette method to screen target sites for selectively silencing AT_1a or AT_1b receptor subtypes in cultured Neuro-2a cells using real-time RT-PCR. For in vivo functional studies, we used C57BL mice with arterial telemetric probes and computerized licking monitors to test the effect of adenovirus carrying the DNA sequence coding AT_1a shRNA (Ad-AT_1a-shRNA). Ad-AT_1a-shRNA was injected into the lateral ventricle (intracerebroventricular) or the brain stem nucleus tractus solitaries/dorsal vagal nucleus (NTS/DVN) with measurement of water intake, blood pressure (BP), and heart rate (HR) for up to 20 days after injection. Tissue culture studies verified the specificity and the efficiency of the constructs. In animal studies, -galactosidase staining and Ang receptor binding assays showed expression of shRNA and downregulation of Ang AT₁ receptors in the subfornical organ and NTS/DVN by >70%. Intracerebroventricular injection of Ad-AT_1a-shRNA increased water intake with no effect on BP or HR. In contrast, microinjection of Ad-AT_1a-shRNA into NTS/DVN caused a decrease in BP with no effect on HR or water intake. Results demonstrate the use of the RNA interference method in site-directed silencing of gene expression and provide a method for the in vivo study of Ang AT₁ receptor function.

Key words: blood pressure • brain • gene regulation • renin-angiotensin system

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