Construction Of Tet-on Lentivirus-mediated Short Hairpin RNA Vector Targeting Human Mirk Gene And Identification Of RNAi Efficiency

[Objective]: To construct Tet-on Lentivirus-mediated short hairpin RNA vector targetinghuman Mirk gene and detect its effect of gene silence in rhabdomyosarcomas RD cells.[Methods]: The specific shRNA sequences targeting human Mirk gene were designed andsynthesized, then linked to Tet-on Lentivirus expression vectors pTRIPZ, after colonyscreening the valid shRNA vectors, the constructed recombinant plasmidpTRIPZ-Mirk-shRNA were identified by digestion with Xhol and Mlul and DNAsequencing. Lentivirus particles were produced with mixed packaging plasmids andrecombinant plasmids co-transfecting to293T cells by calcium phosphate-DNAcoprecipitation, then the acquired lentivirus particles infected rhabdomyosarcomas RD cells,general PCR was performed to determine the mRNA expression level of Mirk.[Results]: Digestion and DNA sequencing results revealed that Mirk-shRNA plasmids werecorrectly constructed, The Lentivirus with a titer of5x106TU/ml was successfullypackaged. When DOX was present, the Mirk expressions in RD cells were obviouslydown-regulated at mRNA level by virus infection compared with controls, the differencewas statistically significant.[Conclusion]: The recombinant Tet-on lentiviral shRNA expression vectors targetinghuman Mirk gene have been successfully constructed. Mirk mRNA can be down-regulatedavailably with DOX induced in rhabdomyosarcomas RD cells.