shRNAs mediate the <i>in vitro</i> allele-specific suppression of mutant or wild-type human ataxin-3 by RNAi.

<p>A–F) shAtaxMUT- or shAtaxWT-encoding plasmids selectively targeting mutant ataxin-3 and wild-type ataxin-3, respectively, resulted in much lower levels of these proteins than the mistargeted control (shGFP) or the non allele-specific shRNA. Quantitative real-time PCR analysis showing the silencing of human ATX3 mRNA in 293T cells co-expressing mutant human ataxin-3 (MUT ATX3) (A, top left) or wild-type human ataxin-3 (WT ATX3) (B, top right) and shAtaxWT, shAtaxMUT, or shGFP. Endogenous ß-actin mRNA was used as an internal control for the normalization and quantitative analysis of the ataxin-3 mRNA levels. Results are expressed as the mean elative mRNA level±SEM. C and D) Western-blot analysis of lysates of 293T cells co-transfected with the plasmid constructs encoding MUT ATX3 (C, middle left) or WT ATX3 (D, middle right) and the shAtax vectors (48 hours after calcium phosphate-mediated transfection; ratio ATX3/shRNA 1∶5). Tubulin staining is shown as a loading control. E and F) Optical densitometry was normalized according to the amount of tubulin loaded in the corresponding lane. A partition ratio was calculated and expressed as a percentage (bottom). All western blots and RT-PCRs shown are representative of three or four independent experiments. Statistical significance was evaluated using Fisher's test (*p<0.05).