Fig. 4.

Disease allele-specific RNAi knockdown against the mutant HTT alleles carrying distinct cSNP haplotypes. (A) ASP-RNAi knockdown by siRs099_T10 and siRs099_C9(G14) siRNAs, which targeted the T and C alleles, respectively, at the rs363099 cSNP site in HTT were examined by IC50 analysis: the effects of the designed siRNAs on suppression of target alleles and allele discrimination were investigated. Data are the average of four independent determinations. Error bars represent SDs. (B) Western blot analyses of the endogenous HTT protein. On the basis of the C0142 and C0221 cSNP haplotypes (Fig. 2C), appropriate siRNA targeting the rs363099 cSNP site in the respective mutant HTT alleles was chosen and introduced into lymphoblastoid cells from the corresponding HD patient. After 3-d incubation, cell lysate was prepared and examined by Western blotting with anti-human HTT antibody; anti-α-tubulin antibody was used as an internal loading control. The siRNAs and lymphoblastoid cell lines are indicated above each gel. A healthy individual (N0001) was also examined as a control. The mutant and normal HTT isoforms are indicated by M and N, respectively.