小鼠转基因研究方法ppt课件

Transgeneandgeneknockoutinstudyinggenefunctions,1,功能缺失(Lossoffunction)：KO,CKO,RNAi获得功能(Gainoffunction)：Transgenic,virus-based在体invivo与离体invitro描述性报告vs机理研究报告研究设计：假说(可能性)验证总结(论文),研究基因功能的两个策略,2,小鼠基因敲除和转基因,这两项技术是用来制备目的基因失活和表达的小鼠。优势：在体研究基因的功能。应用和意义：使得遗传学、肿瘤学、免疫学、发育生物学等学科取得了惊人的进步，这些研究潜在的商业性价值不可估量。,3,基因敲除（knockout）和基因敲入（knockin）KO过程也称基因打靶(genetargeting)，利用DNA同源重组，在基因组中的某一特定部位进行定点的基因置换，用无功能基因替换目的基因或Delete目的基因。根据需要（如验证A与B在功能上有某种关联），在敲除基因A时，将有功能的基因B放在A基因的位置，即A是KO，B是Knockin。,4,基因敲除的基本程序,1、构建打靶载体Targetingvector1）同源序列Homologousarms2）打靶载体常含两种筛选标志,5,positiveselectionneor：新霉素抗性基因，neor基因存在于打靶载体内，当重组（同源或随机整合）后，ES细胞能在含新霉素G418的培养液中生长negativeselection1.HSV-tk：单纯疱疹病毒的胸腺嘧啶激酶基因，该基因产物可分解单核苷酸类似物(ganciclovir)生成毒性产物，产生自杀效果同源重组时：Tk-基因丢失，ES细胞存活随机整合时：Tk-基因存在，ES细胞死亡2.DTA(diphtheriafragmentA):不用给药,6,2、将载体导入ES细胞,Targetingvectorintroducedbyelectroporation,Targetinggene,7,Homologousrecombination,8,Recombinantswithrandominsertion-containingbothneoandtk,Selectionforneorpositive,SelectionforTKnegative,EScellswithhomologousrecombination,ESCells,3.筛选进行了同源重组的ES细胞,9,4、将基因敲除ES细胞注射入囊胚，形成嵌合胚胎，得到嵌合体小鼠，而后筛选培育阳性子代小鼠。,10,ConventionalKnockout,11,MutationofLmx1binmice,Inforelimb,dorsalfeature(hairfollicles)isreplacedbyventralone(footpads).,(Chenetal.,Nat.Genetics,1998),12,Lmx1bisessentialfordevelopmentof5-HT+neurons,(Dingetal.,Nat.Neurosci.,2003),13,Conditionalknockout(CKO)(Tissue-orcelltype-specific),条件Floxedmice目的基因（A）被两个LoxP位点包绕Cremice转基因小鼠，Cre在特定细胞内表达同时表达目的基因（A）和Cre的细胞内的A基因被敲除，其他细胞表达的A基因不受影响,14,Raphenuclei-specificdeletionofLmx1b,(Daietal.,PNAS,2008),(functionalCreexaminationwithRosa26reportermice),15,Mating,viable,16,CKOmiceloseessentiallyall5-HTergicneuronsinthebrain,17,Time-controlCKO,(Daietal.,JCompNeurol,2008),18,GenerationofPet1-CreERmice,(Songetal.,PlosOne),CreER位于胞浆内，不具有活性；在tamoxifen存在情况下，转位入核发挥作用。,19,Lmx1bisrequiredfor5-HTexpressioninAdultBrain,5-HTimmunostaining,tph2immunostaining,20,OthermethodsingeneKnockout,ENU-basedmutagenesisN-ethyl-N-nitrosourea(ENU)wasdevelopedasanefficientmutagenthatwouldcausepointmutationsandsmalldeletionsinmice.IteasytodesigncloselyspacedDNAmarkersformappingofmutations.,21,TheClusteredRegularlyInterspacedShortPalindromicRepeats(CRISPR):RNA-GuidedEndonucleasetechnologyforgenomeengineering.Twocomponents:(1)aguideRNA(2)anendonuclease,CRISPRassociated(Cas)nuclease,Cas9.,/CRISPR/guide/,22,Transgene,23,Transgene,Principles:1.whichtypeofcellsyouareinterested(e.g.isletcells,dopaminergicneurons)2.whatgeneyouwanttoexpressMakingvectors1.Knownpromotorandenhancer2.UnknowncasesBAC-basedmethod,24,Foundermice:transgenicmicethatdevelopfromtheinjectedeggsTransgenicmice:micethataredeterminedtopresenceoftheinjectedgeneStableline:duetomultipleinsertion,25,美国TheJacksonLaboratory世界最大的小鼠品系仓库,26,南京国家小鼠资源中心,27,资源库坐落于南京市浦口高新区，占地100亩，建筑面积7800平方米，小鼠笼位3万多个，各项指标已通过省实验动物环境与设施的监测。引进和自主构建小鼠品系达150多种。,南京国家小鼠资源中心,28,SPF饲养要求,Specificpathogenfree-SPF饲养环境和动物没有特定的病原感染动物福利：环境、对待动物的方式使用量：最小化为原则,29,MouseCare:BarrierAccommodationwithIndividuallyVentilatedCageRacks,空气过滤（万级）、控温和控湿动物防护：更衣、口罩、手套、头套,30,子宫内胚胎电转技术inuteroelectroporation,31,功能缺失（LOF）：KO,CKO,RNAi获得功能（GOF）：Transgenic,virus-based不利的因素:Labor-andtime-consumingSpecialistandequipmentareneeded,研究分子功能的两个策略,32,在体电转技术，可以在任何组织，任何时期（发育和成年）进行LOF和GOF实验LOF：RNAi(virus-based,orvector-based)GOF:Over-expressingwhatyouwant依赖于稳定可靠的在体电转仪的出现,33,Invivo电转的原理,电转后瞬间细胞膜上被击出的孔,带电的质粒在电场作用下胞内的形式,34,Vectors,LOF(RNAi)：pSuperGOF:pCAGGS，强大启动子，表达时间长,35,子宫内胚胎电转InUteroElectroporation,动物:鸡胚，小鼠，大鼠胎龄：鸡胚：越小越容易小鼠、大鼠：越大越容易部位：神经系统、肌肉、胃肠道、皮肤、心脏肾脏、眼睛等等,36,实例一神经细胞迁移,DingYQetal.,Development,2005,37,MisexpressionofDCCinducesventralmigrationofspinalneurons,DingYQetal.,Development,2005,38,DingYQetal.,Development,2004,实例二,39,Knockdown-RNAi,RNAirevealsthatdoublecortinisrequiredforradialmigrationinratneocortex.NatNeurosci.20036:1277-83,实例三,40,如何开始?,几个原则:在水或中性的缓冲液内质粒是带负电的质粒在电场的作用下可以进入细胞内质粒必须在目的细胞的周围BTXhasaproduct(ECM830)thatisabletodothisjobefficiently(),41,Misexpressionvector:pCAGGGSRNAivector:pSuperInjectionvectorsintobrainventricleElectricpulsesPutembryosbackFordetailedprocedures,pleasereadthepaperinDevBio,240:237,2003,byTSaito.,42,电转后GFP在大脑皮层的表达,SaitoBlue-ECFP;Red-DsRed,49,UseTet-onorTet-offsystemtotemporallycontrolexpressionofgenesofinterest.UseCre-Loxpsystemtotransientlyexpressgenesofinterest.,(Mizutami&Saito,Development,2005),适时表达，适时中止,50,鸡胚电转,MorewidelyusedEasytoaccessEggsarecheapforbothGOFandLOF,51,(Odanietal.,Development,GrowthandDifferentiation,200,Vol6),52,53,(Xieetal.,NatCellBiol,2005),Over-expressdominant-negativeprotein,54,Knockdownofgeneexpressioncausesabnormalcelldeath,55,Misexpressionofthreegenesinducesectopicallyexpressionof5-HTinspinalcord,(ChengLetal.,JNeurosci.,2003),56,鸡胚心脏内过度表达基因,57,成年小鼠肌肉内过度表达基因,(Miyazakietal.,Development,GrowthandDifferentiation,2008,Vol6),58,小鼠视网膜内过度表达基因,59,(ZhengMH,etal.,MolBrain,2009),60,小鼠肾脏内过度表达基因,61,大鼠睾丸电转,(Yomgogida,Development,GrowthandDifferentiation,200,Vol6),62,电转mRNA至Xenopus,63,体外培养组织,没有任何技术问题脑片电转后培养单细胞局部少量细胞,(Uesakaetal.,Development,GrowthandDifferentiation,2008,Vol6),64,SummaryI,Explorepossiblefunctionofanewgene/proteinduringdevelopmentaswellasinadult优点:EasytoperforminalabTime-andlabor-savingNotexpensivetosetup,65,Disadvantages:Onlyasmallnumberofne