Construction and identification of double interference vector targeting both PTTG and survivin gene

Objective To construct the double interference vector targeting both PTTG and survivin gene and detect its silencing effects on PTTG and survivin gene in human glioma U251 cell line.Methods Two effective targeting sequences were chosen according to PTTG and survivin cDNA sequences in GenBank,then they were inserted into pGenesil-2.1 vector and pGenesil-2.1-PTTG and pGenesil-2.1-survivin plasmids were constructed.The recombinant plasmids were identified by restriction endonuclease and DNA sequencing.pGenesil-2.1-survivin and pGenesil-2.1-PTTG plasmids were digested by HindⅢ and BamHⅠ separately,long fragment of pGenesil-2.1-survivin plasmid and small fragment of pGenesil-2.1-PTTG(about 380 bp) were collected by 1% agarose gel electrophoresis.The double interference vector pGenesil-2.1-PTTG-survivin siRNA was constructed by uniting long fragment of pGenesil-2.1-srvivin plasmid and small fragment of pGenesil-2.1-PTTG.This plasmid was identified by restriction enzyme digestion and then its silencing effects on PTTG and surviving gene were detected.Results By restriction endonuclease and DNA sequencing,the eukyaryotic expression plasmid of pGenesil-2.1-PTTG,pGenesil-2.1-survivin and pGenesil-2.1-PTTG-survivin siRNA were constructed correctly.The result of RT-PCR showed that PTTG and survivin gene mRNA levels were decreased obviously after this plasmid was transfected into U251 cells for 48 h.Conclusion pGenesil-2.1-PTTG-survivin siRNA plasmid is constructed successfully.The PTTG and survivin gene mRNA levels in U251 cells are decreased effectively by transfecting this plasmid into U251 cells.