The GPA Gene Antisense Nucleic Acid Targets Excavation And Antisense Therapeutic Study

Objective:Screening the targets on pre-mRNAand mRNAof GPAgene and analysis theeffect of each target block gene expression.By compare the interventional effect of targets onpre-mRNA and mRNA for the antisense technology provide guidance and newmethod.Methods:From the K562cells extract the GPAgene and the total RNA.Using mRNAreverse transcribed to cDNA.For the correct sequence,we construct the clone.Using theextra-transcription procure the pre-mRNA and mRNA.Obtaining the targets ofGPA-pre-mRNA and GPA-mRNA by used the MAST method.Doing the verification by theantisense nucleic acid in vitro.Specific-target design the Ribozyme and build the lentiviralvector plasmid.Transfected into K562cells after packaging the lentivirus.Analysis theinterventional effectiveness to GPA gene from the level of mRNA:We have got the positiveclone of GPA gene DNA and cDNA.By transcription,we got the pre-mRNA and mRNA.Conirmed nine potential targets on GPA gene by MAST and verified three availabletargets,determined four positions of the target to intervention.Thereby,we designed theRibozyme to these targets and constructed Lentivirus to transfected K562cells.From theresults of qPCR show that the KD2,KD3and KD4have conspicuously decreased on thelevel of mRNA expression(P<0.05).Obviously,KD4is the best target in every group that it isall above70%on gene knockout.Conclusion:In the experiment,we have got four effectivetargets on the full-length GPA gene.By the technique of Ribozyme find that the one and thethree targets in intron have lower interventional effectiveness than the two and the fourtargets in exon.Moreover,the four is the best target.Accordingly,the antisense target based onthe pre-mRNA don’t have conspicuous significance on gene intervention,mRNA is still thefocus of attention and concern on gene intervention.