動物轉(zhuǎn)基因克隆的分子生物學基礎(chǔ)

動物轉(zhuǎn)基因克隆的分子生物學基礎(chǔ)第1頁/共118頁中國農(nóng)業(yè)大學動物轉(zhuǎn)基因與克隆的研究進展李寧第2頁/共118頁

超級鼠

轉(zhuǎn)GH基因的鼠第3頁/共118頁?4?異種器官移植乳腺生物反應(yīng)器轉(zhuǎn)基因育種血液器官藥用蛋白生長羊毛營養(yǎng)保健HemoglobinDNXBiotherapeuticsIncHeartImutranLimitedKidneyNextrana1-AntitrypinPPLTherapeuticalsplcAntithrombinIIIGenzymeTransgenicCorpLactoferrinPharmingB.V.GlycoconjugatesRossProductsCorpGH,IGF-IPigBreeding,BresgenLtdMP,IGF-ISheepBreeding,AgRes.TRANSGENICANIMALSWhereIsthePotentialforApplication第4頁/共118頁?5?轉(zhuǎn)基因動物研究的里程碑

196619771980-19811981198219831985198719891994199719981999配子的微注射技術(shù)建立mRNA和DNA轉(zhuǎn)移爪蟾卵細胞轉(zhuǎn)基因小鼠獲得成功小鼠胚胎干細胞轉(zhuǎn)移轉(zhuǎn)基因“超級鼠”轉(zhuǎn)基因小鼠組織特異性表達轉(zhuǎn)基因家畜嵌合體“敲除”鼠成功定位整合的種系嵌合體小鼠精子細胞的移植綿羊的體細胞克隆利用核移植技術(shù)產(chǎn)生轉(zhuǎn)基因綿羊“敲除”家畜獲得成功和“Doogie”

鼠第5頁/共118頁?6?Robert(Bob)Wall,KeyPlayerinUSAKeithCampbell,KeyPlayerinUK第6頁/共118頁?7?轉(zhuǎn)基因動物的制作技術(shù)原核期胚胎的顯微注射逆轉(zhuǎn)錄病毒載體注射MII期的卵母細胞精子頭與外源DNA合并注射卵母細胞體細胞核移植技術(shù)制作轉(zhuǎn)基因動物逆轉(zhuǎn)錄病毒載體感染發(fā)育早期的動物胚胎原始生殖細胞技術(shù)精子載體方法胚胎干細胞技術(shù)

第7頁/共118頁?8?MicroinjectionManipulationFacility第8頁/共118頁?9?利用轉(zhuǎn)基因動物制藥的途徑血液、尿腺、乳腺、禽卵、昆蟲120億美元\年的全球市場、20億美元\年藥用蛋白專利過期第9頁/共118頁?10?StrategyforProductionofHumanMedicinebyTransgenicAnimals第10頁/共118頁?11?轉(zhuǎn)基因奶牛和細胞培養(yǎng)技術(shù)生產(chǎn)抗體蛋白的成本比較美國商業(yè)交流公司、Genzyme公司和D&MD醫(yī)藥投資發(fā)展公司報告

第11頁/共118頁?12?美國商業(yè)交流公司、Genzyme公司和D&MD醫(yī)藥投資發(fā)展公司報告

動物乳腺生物反應(yīng)器生產(chǎn)人用藥物蛋白的現(xiàn)狀第12頁/共118頁?13?InterNutria報告、NatureBiotechnology16（8）：728～731，1998

動物乳腺反應(yīng)器生產(chǎn)人醫(yī)用營養(yǎng)保健蛋白的現(xiàn)狀第13頁/共118頁

“人乳化”牛奶是最好的醫(yī)用營養(yǎng)保健品第14頁/共118頁?15?人乳與牛乳主要成分的比較HumanmilkBovinemilkCarbohydraresLactoseOligosaccharidesProteinsCaseinsa-LactalbuminLactoferrinSecretoryIgALysozymeb-lactoglobulinLipidsTriglyceridesPhospholipidsMineralsSodiumPotassiumChlorideCalciumMagnesium73g/l12g/l40g/l1.0g/l3.0g/l1.8g/l1.5g/l1.4g/l1.2g/lnone28g/l1.2g/lTrace0.03g/l0.03g/l3.6g/l4.0%0.04%4.0%0.04%5.0mM

15mM15mM8.0mM

1.4mM15mM

45mM35mM30mM4.0mM第15頁/共118頁?16?人奶與牛奶的功能差異及其優(yōu)缺點Casein:wheyprotein30:7080:20Problems:hardcurdinthestomachanddietaryproteinallergyCysteine:methioninehighlowPhenylalanine&tyrosinelowhighProblems:retardationofbraindevelopmentandPKUbabiesHUMANCOWCalcium:phosphate2.3:11.26:1Problems:hypocalcemia,tetanyandhyperphosphatemia第16頁/共118頁?17?PersonalBallisticProtection:“superiorenergy-to-break”

mechanicalpropertieswithweight25%

lighterthanaramid-basedfibersMedicalMaterials:highstrengthsuturesandprostheticdevicesPersonalHealthcare:cosmeticformulationtoprovidesupportandelasticitytoskinIndustrialComponent:improvethestrengthprofileforautomotiveandmilitaryandaerospacecomponents“生物鋼”材料的應(yīng)用領(lǐng)域第17頁/共118頁?18?“生物鋼”的應(yīng)用領(lǐng)域與發(fā)展需求國防用“生物鋼”-新型生物材料的生產(chǎn)醫(yī)用人工韌帶、關(guān)節(jié)等生物材料的生產(chǎn)高附加值特殊用途-粘性蛋白等的生產(chǎn)歐美市場估計為150億美元/年中國市場估計為100以人民幣/年第18頁/共118頁?19?轉(zhuǎn)基因奶羊和細胞培養(yǎng)技術(shù)生產(chǎn)抗體蛋白的成本比較第19頁/共118頁?20?TheComparisonbetweenDifferentFermentationTechnologyCellCultureMethodsComplexProteinProductionLargeScaleProductionLowCostProductionBacteriaYeastMammalianTransgenicNoNoYesYesYesYesNoYesYes/NoYes/NoNoYes第20頁/共118頁?21?美國商業(yè)交流公司、Genzyme公司和D&MD醫(yī)藥投資發(fā)展公司報告

第21頁/共118頁?22?Time&FinancialRequiredforDevelopmentofTherapeuticalsbyTransgeniccattle01100300550700FeasibilityFounderTransgenicalsTransgenicbreedingProcessdevelopmentCommercialmanufactureClinicaltrialsMarketing01234567891011第22頁/共118頁?23?GenzymeTransgenicCorpFoundin1992,andthefarmwasConstructedin1995.ThetotalEmployeeis672peoplebutonly112ofthemcloselyworkinginThetransgenicplatform.TheallOthersareinapharmacologyDivision.TheNASDAQpriceperShareisaround18-68US$.第23頁/共118頁?24?GoatBarninGenzymeTransgenicCorp第24頁/共118頁?25?TheSituationinsidetheGoatBarn第25頁/共118頁?26?ThedemoGAPbarnfortransgenicmilkinggoat第26頁/共118頁?27?TransgenicGoatswithaSensorLabel第27頁/共118頁?28?MilkingFacilitiesforTransgenicGoats第28頁/共118頁?29?PURIFICATONFACILITIES第29頁/共118頁PPLTherapeuticsplc第30頁/共118頁?31?China“863”High-TechProgramZ21-04ProductionoftherapeuticalproteinsDevelopment1.CAU2.SMI3.MMI4.NGC5.XJIResearch1.CAU2.MAU3.HIU4.GXU5.SBI6.VRI第31頁/共118頁?32?TransgenicDairyGoatwithHumanmATTGene第32頁/共118頁?33?TransgenicDairyGoatwithHumanmATTGene第33頁/共118頁?34?生產(chǎn)人營養(yǎng)保?。ㄡt(yī)療）品！“一種藥物需要10年以上的開發(fā)時間和至少2.5億美元的資金才有可能走向市場，而營養(yǎng)醫(yī)用品只需要幾百萬美元和幾年的時間。更重要的是，營養(yǎng)醫(yī)用品的利潤也非常大”

Dr.LewisLepene，

InterNutria公司執(zhí)行總裁第34頁/共118頁?35?InterNutria報告、NatureBiotechnology16（8）：728～731，1998

第35頁/共118頁ProductiontheHuman-LikeMilkbyState-of-the-ArtTechnology第36頁/共118頁ProgramfortheProductionofNutraceuticalsbyTransgenicCowGenecassetteMicroinjectionTransfectionSelectionNTTransferNewbornsMarket第37頁/共118頁?38?人奶基因基因轉(zhuǎn)染藥物篩選懷孕產(chǎn)犢核卵融合胚胎移植克隆胚胎繁育擴群轉(zhuǎn)基因克隆牛代孕母親工廠化生產(chǎn)牛群市場銷售全球市場營銷體細胞核移植細胞培養(yǎng)第38頁/共118頁ExpansionoftheFounderTransgenicsA.I.&Selection第39頁/共118頁?40?Time&FinancialRequiredforDevelopmentofNutraceuticalsbyTransgeniccattle

FeasibilityFounder&BreedingTransgenicalsTrialsMarketing0123401304050第40頁/共118頁?41?ComparisonofMilkComponent

betweenHumanandCowHumanmilkBovinemilkCarbohydraresLactoseOligosaccharidesProteinsCaseinsa-LactalbuminLactoferrinSecretoryIgAb-lactoglobulinLipidsTriglyceridesPhospholipidsMineralsSodiumPotassiumChlorideCalciumMagnesium73g/l12g/l40g/l1.0g/l3.0g/l1.8g/l1.5g/l1.4g/lnone28g/l1.2g/lTrace0.03g/l3.6g/l4.0%0.04%4.0%0.04%5.0mM

15mM15mM8.0mM

1.4mM15mM

45mM35mM30mM4.0mM第41頁/共118頁?42?ProblemsPresentedbyCow-MilkFeedingInfantsorHumanCasein:wheyprotein30:7080:20Problems:hardcurdinthestomachanddietaryproteinallergyCysteine:methioninehighlowPhenylalanine&tyrosinelowhighProblems:retardationofbraindevelopmentandPKUbabiesHUMANCOWCalcium:phosphate2.3:11.26:1Problems:hypocalcemia,tetanyandhyperphosphatemia第42頁/共118頁?43?MedicalFunctionofHumanLactoferrinBacteriostaticeffectonmicro-organismsBacteriolyticeffectonbroadspectrumofbacteriaGrowthstimulatingeffectonbifidusstrainsIrontransportproteinZinctransportproteinTrophiceffectonmucosalcells第43頁/共118頁?44?PossibleApplicationofHumanLactoferrinasTherapeuticalhLF/hLZasanti-infectiousfactorsforpeopleatriskofgastric&intestinalinfectione.g.younginfants,elderlypeople,patientswithcompromisedimmunologicalresistancehLFasironcarrierforanaemicpeoplee.g.pregnantwomen,elderlypeople,anaemicpatients第44頁/共118頁?45?MoreLactoproteinsinDevelopment

LactoperoxidaseAntiorcytotoxiceffecttopathogeniccellsProlactinImprovementofimmuno-responseZinc-transporterProtectionofstuntedgrowth,alopecia，DermatitisandzinctransportationGrowthfactorsCloseoftheleakymucosalliningofnewbornInhibitionfactorsDirectlysisofinfectedcells第45頁/共118頁?46?MoreLactoproteinsinDevelopment

Aldolase

treatmentofhereditaryfructoseintoleranceCatalase

endothelialrecoveryandagingprocessSuperoxidedismutase

skinandorganinadversityLipoproteinlipase

differenthyperlipidemicdisordersPeroxidase

againstperoxynitrite-mediatedoxidationXanthineoxidoreductase

cardiocirculatorydiseaseGalactosyltransferase

resistanttoparasite&bacteria第46頁/共118頁?47?影響轉(zhuǎn)基因動物產(chǎn)業(yè)化的根本原因落后的轉(zhuǎn)基因技術(shù)-微注射1、轉(zhuǎn)移效率極低(<1%)2、表達效率極低(<1g/l)3、不能定點整合定點進行基因組修飾必將帶來轉(zhuǎn)基因動物產(chǎn)業(yè)化的革命！第47頁/共118頁?48?基因懷孕產(chǎn)犢胚胎移植轉(zhuǎn)基因牛代孕母親表達檢測分子篩選微注射胚胎培養(yǎng)隨機整合傳統(tǒng)微注射技術(shù)生產(chǎn)轉(zhuǎn)基因動物示意圖總效率0.5%第48頁/共118頁?49?核供體動物體細胞細胞培養(yǎng)卵供體動物未受精卵除去卵核細胞融合克隆胚胎胚胎移植無核卵子代孕母親代孕母親與核供體遺傳相同的克隆牛動物體細胞克隆提供解決方案？第49頁/共118頁?50?核供體動物體細胞細胞培養(yǎng)卵供體動物未受精卵除去卵核細胞融合克隆胚胎胚胎移植無核卵子代孕母親代孕母親基因組經(jīng)過隨機整合修飾的轉(zhuǎn)基因克隆牛細胞篩選基因整合陽性細胞擴群目標基因基因轉(zhuǎn)移隨機整合轉(zhuǎn)基因克隆提高了轉(zhuǎn)基因效率但未解決表達效率和時空表達第50頁/共118頁?51?依賴于基因同源重組的轉(zhuǎn)基因克隆可以同時提高基因整合和表達效率核供體動物體細胞細胞培養(yǎng)卵供體動物未受精卵除去卵核細胞融合克隆胚胎胚胎移植無核卵子代孕母親代孕母親基因組經(jīng)過同源重組修飾的轉(zhuǎn)基因克隆牛細胞篩選基因整合目標基因基因轉(zhuǎn)移同源重組第51頁/共118頁?52?同源重組進行基因整合的機理ABNeoRPKillingBAABNeoRP雙交換內(nèi)源性酪蛋白基因突變的酪蛋白基因外源基因結(jié)構(gòu)抗新霉素的細胞系（群）第52頁/共118頁?53?目標基因基因轉(zhuǎn)染藥物篩選懷孕產(chǎn)犢核卵融合胚胎移植克隆胚胎繁育擴群轉(zhuǎn)基因克隆?；蚋咝П磉_代孕母親工廠化生產(chǎn)牛群市場銷售全球市場營銷體細胞核移植細胞培養(yǎng)同源重組依賴于基因組修飾的轉(zhuǎn)基因克隆技術(shù)圖示第53頁/共118頁?54?ProductionofTransgenicAnimalviaCloning第54頁/共118頁?55?Knock-outTechnology第55頁/共118頁?56?HOMOLOGOUSRECOMBINATIONABNeoRPKillingBAABNeoRPDoublecrossoverEndogenouslocusMutatedlocusForeignDNANeomycinresistantcloningcellline第56頁/共118頁?57?HOMOLOGOUSRECOMBINATIONABNeoRPKillingBAABNeoRPSinglecrossoverEndogenouslocusMutatedlocusForeignDNACellswillbekilledbytoxingene’sproductKillingBAEndogenouslocus第57頁/共118頁?58?InducibleexpressionsystemTet-onorTet-offX第58頁/共118頁?59?Cre-LoxPsystemLoxPLoxPCreProteinLoxP第59頁/共118頁?60?IntegrationofmultiplecopiesRNA-mediatedDNAmethylationInsertionintohypermethylatedGenomicregionsTransgenerecognitionDNApairingDNA-RNApairing“Spreading”ofMethylationpatternAgeEnvironmentalstress第60頁/共118頁?61?Howtoovercome“positionaleffect”ChromosomeDNAloopNuclearmatrixchannelActivegenesattachedtomatrixchannelsthroughtheregulatorydomainsandDNApolIIMatrixAttachmentRegion第61頁/共118頁?62?ABCDELCRABCDELocusALocusBLocusCLocusDInsulatorInsulatorLocusControlRegionInsulator第62頁/共118頁?63?AnimalTransgenicsMammarybioreactorPharmaceuticalsNeutraceuticalsBio-materialsBio-cosmetics第63頁/共118頁?64?國內(nèi)外研究現(xiàn)狀與趨勢體細胞克隆動物第64頁/共118頁?65?國內(nèi)外研究現(xiàn)狀與趨勢PPL公司Knock-out綿羊第65頁/共118頁?66?PPL公司Knock-out豬第66頁/共118頁?67?核供體動物體細胞細胞培養(yǎng)卵供體動物未受精卵除去卵核細胞融合克隆胚胎胚胎移植無核卵子代孕母親代孕母親基因組經(jīng)過同源重組修飾的轉(zhuǎn)基因克隆牛細胞篩選基因整合目標基因基因轉(zhuǎn)移同源重組利用同源重組進行基因組修飾的難點長期培養(yǎng)第67頁/共118頁?68?研究技術(shù)路線體細胞克隆細胞長期培養(yǎng)細胞克隆篩選基因的同源重組轉(zhuǎn)基因克隆基因組修飾技術(shù)平臺第68頁/共118頁?69?研究方案和內(nèi)容化學去核法以使體細胞克隆操作簡便、提高工作規(guī)模和克隆效率卵供體動物未受精卵第69頁/共118頁?70?研究方案和內(nèi)容雙標記篩選已確定同源重組的細胞克?。ㄈ海┘毎Y選同源重組新霉素熒光GFP克隆胚胎熒光細胞第70頁/共118頁?71?轉(zhuǎn)有人mAAT基因的奶山羊第71頁/共118頁?72?核供體動物體細胞細胞培養(yǎng)遺傳修飾卵供體動物未受精卵除去卵核細胞融合克隆胚胎胚胎移植無核卵子代孕母親代孕母親與核供體遺傳相同的克隆牛第72頁/共118頁?73?AnimalTransgenicsXenotransplantationOrgansGM-cellsTissuesFrogdevelopmentwithouthead???第73頁/共118頁?74?ImmunologicalBarriersHyperacuterejection:Rejectionmediatedbyantibody-comple-mentcomplexeswhichdamagestheendo-thelialcellsofvascularinterityandkillingtheorganwithinminutesAcuterejection:RejectionmediatedbyTcellswhichdamagethebloodvesselsanddestroyingtheorganwithindaysChronicrejection:RejectionmediatedbyacompleximmuneProcess(humoralimmunityandcell-mediatedimmunity)anddamagingtheorganintherangeofmanyyears第74頁/共118頁?75?用于人體器官移植的動物器官

PPLInterim報告和NatureBiotechnology16（3）：224，1998

第75頁/共118頁?76?美國D&MD醫(yī)藥投資發(fā)展公司

美國等待器官移植的病人數(shù)量（1998年）

第76頁/共118頁?77?Porcineendogenousretrovirusmayhurtthehumanwhenpigorganswastransplantedintothehuman.WhetherpigpathogensWillinfecthumanwhoharboringapigorgansremainunknown?第77頁/共118頁治療型克隆技術(shù)將更具有挑戰(zhàn)性和廣泛應(yīng)用前景？固體器官：60億美元\年的美國市場第78頁/共118頁?79?Xenotransplatationpigs

Theskinwasgraftedfromotherspecies第79頁/共118頁?80?ProductionofBiosteelbyTgGoat5mm第80頁/共118頁?81?ThestrongestmaterialsintheWorldSpiderdraglinesilkiswidelyrecognizedasthestrongestmaterialknown第81頁/共118頁?82?PersonalBallisticProtection:“superiorenergy-to-break”

mechanicalpropertieswithweight25%

lighterthanaramid-basedfibersMedicalMaterials:highstrengthsuturesandprostheticdevicesPersonalHealthcare:cosmeticformulationtoprovidesupportandelasticitytoskinIndustrialComponent:improvethestrengthprofileforautomotiveandmilitaryandaerospacecomponentsPotentialApplicationsofBiosteelinFuture第82頁/共118頁?83?SPIDERMAN-USAARMY?第83頁/共118頁?84?USABiosteelProductionBasementPlattsbaerg,NY第84頁/共118頁?85?診斷、治療人類疾病及新藥篩選的轉(zhuǎn)基因動物模型基因過量的表達基因敲除(Knock-out)或敲入(Knock-in)病毒和其他病原的DNA插入誘變318knock-out-inmice47over-expressionmice15virus-inducedmice

18~25億美元/年市場？第85頁/共118頁?86?TheR&DofGlobalBiotechCompany

BasedonTransgenicPlatformTotalNumbersin1999:56(21inUSA&theNorthAmerica,12inEU,13inAsia&PacificIsland)Thenumbersinstockmarket:10(5inUSAandCanada,4inEuropeancountries,1inAustralia.Thepricerangedfrom7to158US$ThecurrentsituationinChina:3+3+2pending?第86頁/共118頁?87?Xeno-medicineCareOxygentherapeuticalsBiopure,Enzoycorp.InsituEScellstherapy

Chiron,BaxterInc.16.7billioninUSAbut5.8billioninbloodtransfusion第87頁/共118頁?88?BIOPURE:acinderalatechnologyHemopure:animmediateoxygendelivermedicineformanydiseases(clinicaltrialIIIfinished)Oxyglobin:anoxygentherapeuticalsforpetanimals第88頁/共118頁?89?BiopureHeadquarterinBostonNASDAQ:22.5US$/pershare.But300millionsspent100millioninhandforheadingtomarket第89頁/共118頁?90?ANIMALCLONING&ESCELLTECHNOLOGY19971997第90頁/共118頁?91?第91頁/共118頁?92?Cloningtechnologyofanimalproposed,1938,SpemannAdultfrogsfromthenuclearofsomaticcells,1962,GurdonNucleartransplantationinsheepembryo,1986,WilladsenProductionofidenticalbovineoffspring,1990,BondioliSheepclonedfromaculturedcellline,1995,CampbellViableoffspringfromadultmammaliancells,1997,WilmutClonedmiceproducedbyinjectedcellnuclei,1998,WakayamaMilestoneofAnimalCloning第92頁/共118頁?93?第93頁/共118頁?94?Micewereclonedinalargescaleby“Honolulu”technologyeggnucleusmotherclonedcloned第94頁/共118頁?95?第95頁/共118頁?96?CollectingtissueandcellculturePetorfarmanimaltobecloned第96頁/共118頁?97?CellsStorageforCloningtorelive第97頁/共118頁?98?Manipulationofrecipientoocytes第98頁/共118頁?99?NUCLEARTRANSFER第99頁/共118頁?100?EmbryoTransfer第100頁/共118頁?101?第101頁/共118頁?102?ClonedtransgeniccalvesproducedfromnonquiescentfetalfibroblastsCMVLacZNeoRTransfectionNeo-resistantNucleartransferEmbryotransferClonedcalves第102頁/共118頁?103?CloningPandaandMammothRareintheworldDeadover12,000yrs第103頁/共118頁?104?Cross-speciesanimalcloning第104頁/共118頁?105?CloningHumanorOrgan?第105頁/共118頁?106?Therapeuticalcloningprocedure第106頁/共118頁?107?Thecellscanbeindu