miRNA-based Gene Silencing Constructs

To generate the miHTT-155 and miSNP67T-155 constructs, their guide sequences targeting HTT transcripts were embedded into the engineered murine pre-miR-155 backbone of pcDNA6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) by annealing complementary oligonucleotides (Qiagen, Valencia, CA, USA) followed by ligation into the linearized pcDNA6.2 plasmid.^{18 (link)}To generate miHTT-451 and miSNP50T-451 constructs, the guide and passenger strand sequences were incorporated in the hsa-pri-miR-451a scaffold. 200 nucleotides long 5′ and 3′ encompassing flanking regions were included with EcoRV and BamHI restriction sites and the complete sequences were ordered from GeneArt gene synthesis (Invitrogen). In these constructs, the cytomegalovirus promoter was replaced by the CAG promoter (Inovio, Plymouth Meeting, PA).^{18 (link)}