哺乳动物细胞的基因工程与转基因动物课件

哺乳动物细胞的基因工程与转基因动物PartI

GeneticEngineeringinMammalianCells哺乳动物细胞的基因工程1.SelectiveMarkersinMammalianCells哺乳动物细胞的基因工程SelectiveMarkersinMammalianCellsHATmedium:Hypoxanthine,Aminopterin,ThymidineHPRT:HypoxanthinephosphoribosyltransferaseTK:Thymidinekinase

哺乳动物细胞的基因工程SelectiveMarkersinMammalianCells

哺乳动物细胞的基因工程ENZYME(abbreviation)DRUGFORSELECTIONSELECTIONMECHANISMAminoglycosidephosfotransferase(APH)G418(inhibitsproteinsynthesis)APHinactivatesG418Dihydrofolatereductase(DHFR):Mtx-resistantvariantMethotrexate(Mtx;inhibitsDHFR)VariantDHFRresistanttoMtxHygromycin-B-phosphotransferase(HPH)Hygromycin-B(inhibitsproteinsynthesis)HPHinactivateshygromycinBThymidinekinase(TK)Aminopterin(inhibitsdenovopurineandthymidylatesynthesis)TKsynthesizesthymidylateXanthine-guaninephosphoribosyltransferase(XGPRT)Mycophenolicacid(inhibitsdenovoGMPsynthesis)XGPRTsynthesizesGMPfromxanthineAdenosinedeaminase(ADA)9-β-D-xylofuranosyladenine(Xyl-A;damagesDNA)ADAinactivatesXyl-ASelectiveMarkers：APHandG418

G418是一种氨基糖苷类抗生素,在基因工程实验中,是稳定转染最常用的抗性筛选试剂。它通过抑制转座子Tn601，Tn5的基因,干扰核糖体功能而阻断蛋白质合成,对原核和真核等细胞产生毒素,包括细菌、酵母、植物和哺乳动物细胞,也包括原生动物和蠕虫。当neo基因被整合进真核细胞DNA后,则能启动neo基因编码的序列转录为mRNA

,从而获得抗性产物氨基糖苷磷酸转移酶的高效表达,使细胞获得抗性而能在含有Ｇ418的选择性培养基中生长。G418的这一选择特性,已在基因转移、基因敲除、抗性筛选以及转基因动物等方面得以广泛应用。哺乳动物细胞的基因工程2.Genetransfera.Calciumphosphatecoprecipitation哺乳动物细胞的基因工程b.CotransfectionTransferofanonselectablegenebycotransfectionwithamarkergeneHSV-Herpessimplexvirus哺乳动物细胞的基因工程c.Electroporation

哺乳动物细胞的基因工程d.lipofectionLiposome-mediatedgenetransfer哺乳动物细胞的基因工程3.GeneexpressionTransfectedDNAismaintainedtransientlyinmanycellsaph-Aminoglycosidephosphotransferase(氨基糖苷磷酸转移酶)哺乳动物细胞的基因工程Atypicalmammalianexpressionvectorforhigh-levelproteinexpression哺乳动物细胞的基因工程

Obtaininghigh-levelexpressionbycoamplificationwithdhfr(dihydrofolatereductase)Mtx-methotrexate哺乳动物细胞的基因工程SV40viralvectors哺乳动物细胞的基因工程UseofCOScellsforhigh-levelproteinexpression哺乳动物细胞的基因工程Expressionofthecysticfibrosis(CF)geneinavacciniavirusvector哺乳动物细胞的基因工程Baculoviruscanbeusedforhigh-levelproteinproduction哺乳动物细胞的基因工程Useofaretrovirusvectorforstable,long-termexpressionofaforeigngeneLTR-LongterminalrepeatUseofantisenseRNAtoextinguishcellulargeneexpression哺乳动物细胞的基因工程4.GenetargetingTwostrategiesforenrichingforgenetargetingorhomologousrecombinationevents哺乳动物细胞的基因工程GCV-ganciclovir丙氧鸟苷UsingPCRtodetectgenetargetingeventswithouttheuseofaselectablemarker哺乳动物细胞的基因工程PartII

GeneticEngineeringinMammalianAnimals—TransgenicAnimals转基因动物1.Microinjectionapproach转基因动物Producingtransgenicmicebymicroinjection转基因动物MicroinjectedforeignDNAcanbetransmittedthroughthegermlineaswithanormalMendeliangene转基因动物InjectSrytransgeneintoXXegg转基因动物2.EScellapproachEmbryonicstemcells(EScells)producedfrommouseblastocysts转基因动物Embryonicstemcells(EScells)producedfrommouseblastocysts

转基因动物转基因动物Constructingofthetargetingvector.(2)Genetargetinginembrio-derivedstem(ES)cells.(3)Selectionofgenetargetedcells.(4)IntroductionoftargetedEScellsintomouseembryosandimplantationintoafostermother.(5)Analysisofchimericmiceandinbreeding.3.TransgenicanimalsGenetargetingtospecifictissues转基因动物Productionofapharmaceuticallyimportantproteininthemilkoftransgenicsheep转基因动物Productionof

“supermouse”转基因动物“Super”mouse.Atransgenicmouse(left)expressingtheratgrowthhormonegeneundercontrolofthemousemetallothioneingenepromotergrewtotwicethesizeofanormalsibling(right).Transgenicartwork:GFPbunny转基因动物Geneticallymodifiedpets:Glofish:transgeniczebrafish,expressingRFP(B)Copycat:Thefirstclonedcat(C)Snuppy:Afghanhound,thefirstcloneddog转基因动物OtherKindsofTransgenicMiceOncomouse

-c-myconcogeneandsectionofthemousemammarytumor(MMT)virusgaverisetobreastcancerbyPhilipLederinHarvardUniversity,1982(thefirstcomplexanimaltobegrantedapatentintheUSA)Prostatemouse-prostatecancerSCIDSmouse-micewithseverecombinedimmunodeficiencysyndromeAlzheimermouse-mousethatshowsymptomsofAlzheimerdisease转基因动物OtherKindsofTransgenicMiceKnockoutmouse-CFKnockoutmouse1995,MKMD,[/mkind/],5000entriesin2002ProteinProductionTransgenicAnimal-tPAtransgenicmice(WAP-tPA),FIXtransgenicsheep(BLG-FIX)转基因动物

转基因动物TransgenicSalmon-20001989年，加拿大研究人员成功在鲑鱼中超量表达生长激素。1995年，转基因鲑鱼的开发商，来自美国马萨诸塞州Maynard的AquaBounty公司向FDA提出新型畜牧用药调查申请。（FDA没有转基因动物的申请类别，故AquaBounty将转基因技术作为畜牧用药。）2001年，AquaBounty公司向FDA提交了第一份调研报告。2009年，FDA发表对转基因技术作为畜牧用药的评估指南，AquaBounty公司向FDA提交了所有需要的文书。2010年，FDA认为转基因鲑鱼可安全食用。2012年，FDA在五月完成了转基因鲑鱼环境影响评估草案，但直到十二月才发布。2013年，环境影响评估草案的公示期被延长了两个月，到4月26日结束。

Geneticallymodifiedsalmon,thefirsttransgenicanimalforfoodproduction转基因动物AquAdvantagesalmon：ageneticallymodifiedAtlanticsalmonGrowthhormonecDNAfromChinooksalmonAntifreezeproteinopAFPfromOceanpoutFemaleAtlanticsalmonAquAdvantagesalmonWildAtlanticsalmon生长激素提高产量大有可为草鱼的GH基因导入鲫鱼转基因动物Transgenicmonkey转基因猕猴

转基因动物TransgenicMonkey(2001)(LEFT)Virusparticlescarryingthegreenfluorescentprotein(GFP)geneareinjectedintotheunfertilizedegg.Thegene(white)isreleasedfromthevirusandincorporatedintothechromosome.

(RIGHT)About6hoursafterintroducingthevirusscientistsartificiallyfertilizetheeggbyinjectingaspermfromamalerhesus.Thefertilizedeggthenbeginstogrowanddivide.Twotothreeday