心肌条件性Pellino1基因敲除小鼠的构建与鉴定#李建涛1，范敏1，夏薇2，阙玲俐1，李跃华1**51015202530354045（1.南京医科大学基础医学院心血管病分子干预重点实验室，南京210029；2.南京医科大学医药动物实验基地，南京210029）摘要：目的：构建心肌条件性Pellino1基因敲除小鼠，并进行鉴定和表型分析。方法：利用同源重组原理构建打靶载体，采用电转导方法将线性化打靶载体转入胚胎干细胞（ES细胞）。将正确同源重组的ES细胞进行囊胚腔注射，并将其移植至代孕小鼠体内，生育嵌合体小鼠；嵌合体小鼠和C57BL/6J鼠交配繁殖出杂合子，再与Myh6-cre/Esr1鼠交配得到Myh6-cre/Esr1阳性的Flox/Flox鼠，采用PCR方法鉴定小鼠的基因型。Myh6-cre/Esr1,Flox/Flox小鼠注射他莫昔芬诱导心肌Pellino1基因敲除。采用小动物超声分析心肌特异性Pellino1敲除小鼠的心功能，HE染色与天狼猩红染色观察心肌组织形态结构的变化。结果：成功构建了Pellino1同源重组载体，经PCR鉴定为阳性；完成Pellino1基因打靶及囊胚注射，经鉴定为阳性；繁育出了Myh6-cre/Esr1阳性，Flox/Flox阳性鼠；在体他莫昔芬诱导心肌Cre表达，产生心肌特异性Pellino1敲除小鼠；与野生型小鼠相比，心肌条件性Pellino1敲除两周对心脏EF值、FS%、IVS、LVPW无明显影响，而且其心肌形态结构与野生型小鼠相比亦未见明显差异。结论：成功构建出心肌条件性Pellino1基因敲除;Pellino1心肌特异性敲除两周对心肌形态结构和心功能无明显影响。关键词：Pellino1；基因敲除；心肌条件性；心功能中图分类号：R363GenerationandidentificationoftheconditionalcardiacPellino1geneknockoutmouseLIJiantao1,FANMin1,XIAWei2,QUELingli1,LIYuehua1(1.Keylaboratoryofcardiovasculardiseaseandmolecularintervention,NanjingMedicalUniversity,Nanjing210029;2.Animalexperimentcenter,NanjingMedicalUniversity,Nanjing210029)Abstract:Objectives:TogenerateinduciblecardiacconditionalPellino1knockoutmouse,andidentifythephenotypeofmousegenerated.Methods:Theconstructoftargetingvectorwasmadebyhomologousrecombination.ThevectorwaslinearizedandelectroporatedintoEScells.HomologousrecombinantswasidentifiedbyPCRafterselectingthestabletransfectants.ThepositiveclonesweretheninjectedintoC57BL/6Jhostblastocysts,andtheinjectedblastocystsweretransferredintopseudopregnantfostermothersinordertogivebirthtochimericmice.ChimericmiceandC57BL/6Jmicethengavebirthtopotentialheterozygotefounders.Matingoftheheterozygotestogethomozygotes.ThegenetypeofthesemicewereassayedbyPCR.GaveTamoxifentoripehomozygotestoinducetheknockoutofPellino1.MeasurethecardiacfunctionandthemorphologyofmyocardialcellsofFlox/Floxmice.Results:HomologousrecombinationvectorofPellino1wasidentifiedaspositivebyPCR;Pellino1genetargetingandblastocystinjectionwereidentifiedaspositive.Esr/Myh6-CreandFlox/FloxpositivemicewereconstructedandinjectionoftamoxifeninducedmyocardialexpressionofCreinvivo,resultingincardiac-conditionalknockoutPellino1mice.PathologicalanalysisandechocardiographyassessmentindicatedthattherewerenosignificantdifferencesbetweenPellino1knockoutmiceandtheirWTcongenersoncardiacfunction.Conclusion：Pellino1genehasbeensuccessfullyknockedout,andinduciblecardiacconditionalPellino1geneknockoutmousehasbeensuccessfullygenerated.knockoutofPellino1hadnoeffectoncardiacfunctionaftertwoweeks.Keywords:Pellino1;geneknockout;cardiacspecific;cardiacfunction基金项目：TIR/BB环拟似物AS-1改善压力超负荷型心力衰竭的作用机制研究（20113234110007）；Pellino1蛋白及其SUMO修饰在糖尿病心肌病中的作用机制研究（NSFC81270292/H0208）作者简介：李建涛，（1985-），男，博士生，心血管疾病与信号转导。通信联系人：李跃华，（1964-），女，教授，心血管疾病与信号转导。yhli@nj...