Construction and identification of survivin-targeting siRNA-expressing plasmid / 临床检验杂志

ABSTRACT

Objective To construct survivin-targeting siRNA-expressing plasmid.Methods DNA sequence correspond to siRNA targeting survivin was designed and synthesized,and cloned into plasmid pRNAT-U6.1/Neo to produce surviving-targeting plasmid.Two oligos in the template with cohesive BamHⅠ and HindⅢ sites were prepared and annealled to form the insert fragment for siRNA vector.The vector was cut with BamHⅠ and HindⅢ and ligated with the insert fragment using T4 ligase.The recombinant vector was confirmed by restriction digestion and DNA sequencing,and then was transfected into T24 cells with Lipofectamine TM2000 and the expression of survivin was detected by real-time quantitive PCR.Results DNA sequencing for the PCR product showed that the recombinant vector pRNAT-U6.1/Neo-survivin was successfully constructed without any base pair mutation.The plasmid pRNAT-U6.1/Neo-survivin could efficiently reduce the expression of survivin and confer G-418 resistance in T24 cells.Conclusion The siRNA-expressing plasmid which were successfully constructed and transfected into T24 cells in this study may facilitate the application of RNA interference technique,and lay foundation for further studies on the function of survivin.