( he brain striatal region of transgenic ...)Superior Gene Silencing Using Adenoviral Vectors

he brain striatal region of transgenic mice expressing GFP. The adenovirus included a dsRed expression cassette to visualize the localization of the virus. (A) Brain sections were analyzed by fluorescence microscopy 5 days after injection. Green: GFP. Red: dsRed. Left panels: GFP detection. Middle panels: dsRed detection. Right panels: merged images. (B) Brain sections were divided into ipsilateral (il) and contralateral (cl) portions and analyzed by Western blot using antibodies to GFP and -actin (control).

Allele-specific Silencing of Dominant Disease Genes
Due to its inherent specificity, a potential application of siRNAs is the treatment of dominantly inherited diseases. One such neurodegenerative disease is Machado-Joseph disease/spino-cerebellar ataxia type 3. In 70% of diseased carriers, alleles contain a G to C mutation. Dr. Davidson's laboratory constructed plasmids fusing GFP to normal (Atx-Q28-GFP) or mutant polyglutamine (polyQ) alleles (Atx-Q166-GFP) containing a CAG expansion in Ataxin-3 (2). Adenoviral vectors that expressed hairpin siRNAs targeting either the wild type or the mutant allele were designed generated from these plasmids and used to infect Cos-7 cells that had previously been transiently co-transfected with Atx-Q28-GFP (wt) and Atx-Q166 (mutant). (Both plasmids were co-transfected into the Cos-7 cells to mimic the heterozygous adenovirus.) The siRNA expressed from AdG10i had perfect complementarity to the wild type allele, and preferentially suppressed expression of the fusion construct containing that allele (Atx-Q28-GFP, Figure 3A). Conversely, AdC10i preferentially suppressed expression of the mutant allele construct (Atx-Q166, Figu
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