Construction And Identification Of Lentiviral Vectors Of IK6-specific ShRNA

Part Ⅰ Designing and Screening of siRNA sequences to IK6geneObjective:To design and synthesis small interfering RNA(siRNA) to IK6,then screen the most efficiency siRNA sequence;construction and identification of lentiviral vector of IK6specific short hairpin RNA(shRNA),which lay the foundation for RNA interference(RNAi) mediated gene therapy of children acute lymphoblastic leukemia(ALL).Methods:Three pairs of siRNA to IK6gene were designed and synthesized, ALL cell line Sup-B15was transiently transfected. After transfection,the growth and morphology of Sup-B15were observed by fluorescence microscopy;transfection efficiency was determined by flow cytometry;Gene and protein expression of IK6were detected by RT-PCR and Western-Blot.According to the result of part one,oligo DNA to target sequence was synthesized and annealed to form double stranded DNA(ds DNA). The expression vector named pLV! shIK6wasconstructed by linking the ds DNA and GV112both cut by AgeI andEcoRI. pLV! shIK6were packaged by co-transfecting293T cells withother two plasmids (pHelper1.0and pHelper2.0).The titre of lentivirusparticle was detected by titration dilution. The Ik6-shRNA lentivirusparticle infected Sup-B15cells and the Ik6expression was detectedby real-time quantitive PCR and Western Blot on the level of mRNA andprotein.Results:Three pairs of siRNA to IK6were successfully designed andsynthesized,then transiently transfect ALL cell line Sup-B15.Aftertransfection,there was less effect on cell growth and morphology;transfection efficiency of Sup-B15can reach up to80%,which met theexperimental requirements;The result of RT-PCR and Western Blotshowed the significant down-regulation of Ik6mRNA and proteinexpression in Sup-B15. The pLV! shIK6was confirmed precisely by PCRand sequencing.The titre of lentivirus particles was8×108TU/ML andmet the demand of the following experiments. When multiple of infection(MOI) was100, the infection efficacy of Sup-B15cells was80%.Theresult of RT-PCR and Western Blot showed the significantdown-regulation on the level of transcription and protein expression ofIK6in Sup-B15after transfection(P﹤0.01).Conclusion: One most efficency pair of siRNA to IK6was determined. The lentivirus vectors of Ik6-shRNA were successfully constructed withhigh infection efficiency and the stable expression of Ik6in Sup-B15cells. It is the experimental foundation for construction of lentivirusvector and further research on the role of Ik6in leukemia.