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. Author manuscript; available in PMC: 2009 Mar 17.
Published in final edited form as: Mol Ther. 2005 Oct;12(4):618–633. doi: 10.1016/j.ymthe.2005.05.006

FIG. 3. Long-term in vivo striatal expression of rAAV5-siHUNT1 and rAAV-siHUNT2 decreases mHtt trans-gene mRNA expression.

FIG. 3

(A) In situ hybridization with a probe against the virally encoded hrGFP mRNA. Coronal sections were obtained 10 weeks postsurgery. The images are arranged on a rostral–caudal axis to illustrate the extent of the transduced area as indicated by the arrow. Two separate injections were placed into the same hemisphere (arrows). These sections were obtained from one animal and are representative of all other study animals. Scale bar, 2 mm. (B) Photomicrograph of native hrGFP protein expression from the striatum of a representative injected animal demonstrates that the vector-derived mRNA shown in (A) is transcribed into protein. STR, striatum; cc, corpus callosum; and CTX, cortex. Scale bar, 1 mm. (C) Real-time quantitative PCR analysis of mHtt mRNA knockdown. Total RNA was extracted from the striata of R6/1 mice. The levels of striatal mHtt mRNA on the uninjected (black bars) striata and striata injected with rAAV5 vectors (white bars) are shown. rAAV5-siHUNT-1, rAAV5-siHUNT-2, and rAAV5 siRho were measured, normalized to the levels of HPRT mRNA, and reported as a percentage of control. Expression of rAAV5-siHUNT-1 resulted in a significant 75% reduction of mHtt mRNA expression (F[1,11] = 40.0, *P < 0.001). Striatal rAAV5-siHUNT-2 treatment resulted in significant 78% reduction of striatal mHtt mRNA expression (F[1.8] = 19.1, *P = 0.002). In contrast, striatal injection of the control shRNA rAAV5-siRho did not reduce striatal mHtt mRNA levels (F[1,8] = 0.71, P > 0.40). (D) Similar to striatal mRNA analysis in (C), total protein was obtained from the injected and untreated control striata of R6/1 mice. Similar to the mRNA levels, there was a significant rAAV5-sitHUNT-1-mediated 24.5% reduction in the levels of a Hum-1-immunoreactive high-molecular-weight band (simple main effects, F[1,18] = 5.2, *P < 0.04). The rAAV5-siHUNT-2-treated group displayed a significant 38% reduction of striatal mHtt (simple main effects, F[1,18] = 8.4, *P = 0.01), while the rAAV-siRho control groups did not have significant reductions of striatal mHtt (simple main effects, F[1,18] = 0.50, P > 0.40) compared to R6/1 control striata. Error bars represent +SEM.