AimTo construct a pSUPER RNAi system that targets human heart developmental candidate gene hole and a stable virus-producing cell line.MethodsA pair of 60nt oligonucleotides coding for short hairpin RNA and targeting human heart developmental candidate gene hole were chemically synthesized and annealed and inserted into pSUPER plasmids digested with BglⅡ and XhoⅠto construct the recombinant pSUPER RNAi plasmid (pSUPER-hole).Recombinant pSUPER-hole plasmid was identified by enzyme digestion and sequencing analysis.The packaging cell H9c2 was transfected with the recombinant plasmid.The mRNA expression level of hole was detected by RT-PCR.ResultsThe result of enzyme digestion and sequencing analysis demonstrated that 60 nt had been inserted successfully into the vector.Green fluorescent was detected in virus-producing cells, RT-PCR showed pSUPER-hole can inhibit the hole gene expression of H9c2. ConclusionThe pSUPER RNAi vector targeting human heart developmental candidate gene hole are successfully constructed.