大鼠聚腺苷二磷酸核糖聚合酶-1基因RNA干扰表达质粒构建与鉴定

《大鼠聚腺苷二磷酸核糖聚合酶-1基因RNA干扰表达质粒构建与鉴定》由会员上传分享，免费在线阅读，更多相关内容在工程资料-天天文库。

1、文章集号4095-2619(2012)01-0001-04大鼠聚腺昔二磷酸核糖聚合酶-1基因RNA干扰表达质粒构建与鉴定高羽辛心，黄明元2,刘林华?，梁海荣蔦唐焕文2摘要：目的构建并筛选大贰聚腺药二续咬换殊聚合基因的RNA干扰(RNAi)表达质炷，为职业惶戎患的基因治疗探索新途轻。方法根坍基因序列数据阵中报道.的PARP-I基因净列及短发夬RNA(shRNA)设计原则，设计、合成用于构建RWAi质粒的眾核并酸，构瑾4聲支姐PARPJRNAi用粒.醉切及測序鉴定正确后•以脂质体U-pofecUminJYOOO介导转染大风骨牡间充质干仙胞。48h后，采用逆转敢聚合聲链反应(RT・P(：R)技术检

2、测輔染细胞中PARPJmRNA的转录水平，觴选有效的PARP-1R¥Ai质粒。结果4种重组PARPJRNAi质粒经酹切及测序证明构建正确"转染后48h,转染质粒shRNA-PARP-1-532的细袍PARP-J基因柯制效果最明星，mRNA的转录水乎下隆了78%,可作为后裟实脸的有效质杜:：＞结论成功构建并捕il出PARP1基因的RNAi表达贞为探索PAKP-I基因在疾病中的作用莫芝了崔础。关镇词：聚腺夺二辑絞根純聚合酶-】；隸发央RNA｛骨戕间充质干佃胞；转棗；大鼠中国分类号：R135文就标识码：AConstructionandscreefiingofRNAiexpressionvector

3、forratpoly(ADP-rib<>Ke)polymerase-!geneGAOYu-ting,HUANGMing-yuan,LIULin-hua,LIANGHai-n)ngtTANGHuan-wcn(DepartmentofToxicologyrSchoolofPublicHealth,JilinUniversity.Changchun130021,China)Abstract:ObjectiveTooonBtructandscreentheRNAiuxpn^onvec

4、eanovelrouteforthegenetherapyofoccupationaldiseases.MethodsTheoligonucleotideswmvdc»igi>rdtukdsynthesizedaccordingtothePARP-1genereportedinGenBankandthegenera]principleofshRNA(1疥ign.basedonwhich4m・combinantRNAiexpressionvcctoratargetingPARISIgenewereconstructedwidentifiedbyrestiictionanalysisandseq

5、uencing,thentransfectedtorathonemesenchymal^temcellsinmediationofUpofecuunine™2000.ThetranscriptionlevelsofPARP-1mKNAweredetenninedbywal・timercverectranscriptionpolymera^chainrcaclion(RT・P(：R)48hoursaftertrarusfretiontoscreenthevalidRNAiexpressionvectorstargetingPARPJgene.ResultsRestrictionanalysis

6、andsequencingprovedthat4recombinantRNAiexpressionvectorsUrgedry;PARP-1genewereconstructedcorrectly.ThetranscriptionlevelofPARP-1mRNAinbonemesenchymalstemcellstransfectedwithrecombinantplasmidsshRNA-PARP-1-532decreasedmoreremarkablr.hwasusedasthevalidRNAiexpressionvectoratargetingPARP-1geneforfurth

7、erstudy.ConclusionTheRNAiexpressionvectorforratPARP-Igenewassuccessfullyconstructedand乂reentd,whichlaidafoundiitionofstudyontheeffectofPARP-1g<«cinthediseases・Keywords：Poly(AI)F,-nlxk€e)p<>lymeraj