Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells / 中华劳动卫生职业病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells.</p><p><b>METHODS</b>Spe I and Xho I-digested insert from pSUPER, which comprised H1 RNA polymerase III promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 (+) to generate pSUPER/neo that could express small interfering RNA in mammalian cells. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl II and Hind III to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay.</p><p><b>RESULTS</b>pSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells could efficiently inhibit the activity of NTE in the mammalian cells.</p><p><b>CONCLUSION</b>Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.</p>