[Construction and detection of gene expression vector of the Von Hippel-Lindau in bone marrow stromal cells]

To construct siRNA-VHL expression vector and detect the effect of VHL gene interference on BMSCs.

According to the dog's VHL gene sequences, four pairs of siRNA oligo were designed and synthesized. Using vector cloning kit reorganization, four pairs of double-stranded siRNA were inserted into the expression vector (pcDNA™ 6.2-GW/EmGFPmiR) and 4 siRNA expression plasmids (SR144-1,SR144-2,SR144-3,SR144-4) were constructed. With the vector universal primers, colony PCR was screened. The positive clones were sequenced to verify whether the sequence of insert fragments in recombinant clones was consistent with oligo sequences designed or not. Interference vector transiently transfected the BMSCs. qPCR and Western blot were used to detect the gene silencing effect. In order to improve transfection efficiency of siRNA-VHL as well as the effect of the VHL gene silencing, pLenti-mi-VHL was constructed.

Through sequencing the plasmids cloned, the fragment sequences inserted in recombinant clones were consistent with the designed oligo sequences. After 24 h and 48 h transfection of BMSCs cells by plasmids, SR144-4 showed the best effect of interference by qPCR and Western blot. Through comparing the sequencing results, the inserted fragment sequences were completely correct and the pLenti-mi-VHL was successfully constructed.

The siRNA-VHL expression vector for BMSCs is successfully constructed and applicable for further experiments. Supported by Research Fund of Science and Technology Commission of Shanghai Municipality (Grant No.9411954800) and Foundation for Open Project from Shanghai Key Laboratory of Stomatology (Grant No.S30206).