Construction and Identification of Lentivirus-mediated Short Hairpin RNA Targeting Human FOXM1 Gene

Objective To construct and identify a lentivirus-mediated short hairpin RNA(shRNA) targeting human FOXM1 gene.Methods The shRNA sequence targeting to human FOXM1 gene was designed and synthesized,then linked to lentivirus expression vector pLL3.7 containing U6 promoter and green fluorescent protein(GFP) gene by gene recombination technique.The constructed recombinant plasmid pLL-FOXM1-shRNA was identified by digestion with XbaⅠand NheⅠand DNA sequencing,then mixed with lentivirus packaging plasmids and co-transfected to 293T cells with LipofectamineTM 2000.Supernatant was collected 72 h after incubation,from which lentivirus particles were purified by high speed centrifugation and determined for titer by hole-by-hole dilution method.Results Both restriction analysis and sequencing proved that shRNA sequence targeting human FOXM1 gene was successfully inserted into vector pLL3.7.Lentivirus particles were successfully packaged in 293T cells,of which the titer was 1 × 108 TU / ml.Conclusion The lentivirus-mediated shRNA targeting human FOXM1 gene was successfully constructed,which laid a foundation of further study on molecular function of FOXM1.