遺傳工程小鼠的制備繁育及應用.(transgenicinsertionalmutantswithobesity)Timo“transgenic”miceStory

1.MouseCh2RegulatorylocusBDNF/BdnfTransgeneinsertionLCRC1RC2RC3Transgeneintegrationsite~7kbSha,H.,etal(,2007)PhysiolGenomics.31(2):p.252-63Timo“transgenic”mice.第一個轉基因小鼠（1982）byRichardPalmiterandRalphBrinster.The“conventional”transgenesisisusingshortconstructswithwell-definedpromoterregions.

ProcedureofconventionalTG.基因組改造技術是建立疾病模型的核心2007年諾貝爾生理醫(yī)學獎CappechiEvansSmithies2006年EUCOMM:1.4億歐元NorCOMM:1億加元KOMP:

1.8億美元ChCOMM:

1500萬RMB第一個基因敲除小鼠（1987-89）.2SAIRESlacZpANeoRpA3465FRTFRTloxPloxPloxP1234651WildtypeAllele261NeoRpAloxPloxP234651loxPNeoRpAFRTloxPFRT234651*NeoRpAloxPloxPKnockoutAlleleConditionalKOAllele(flox)Knock-inKOfirst,ConditionalReadyAlleleEStargetstrategy.PI3K-PKB/AktPathwayinCardiacsystemPKBalphaPKBbetaPKBgamaPP2APDK1(cKO,TM)Story2蛋白磷酸化調控.出生后心臟發(fā)育不良和心衰Yinetal,GenesDev,2012;Zhangetal,J.Pathol.,2012Dietal,Biochem.J.,2012;Luetal,PlosONE,2011Fangetal,MolCellBiol,2010; Chang

etal,DevBiol,2010;Zhouetal,AmJPath,2010; Chang

etal,FrontBiosci,2010心臟ASD(房室隔缺陷).Fstl1,Follistatin-like1中國第一個條件性敲除模型（2005）故事三.nervoussystem.LungDevelopment.UreterDevelopment.TheCRISPRCrazeAbacterialimmunesystemyieldsapotentiallyrevolutionarygenome-editingtechniqueScience

23August2013vol.341NEWSFOCUS

“Idon’tthinkthere’sanyexampleofanyfieldmovingthisfast”----BlakeWiedenheftMontanaStateUniversityTheCRISPRCraze.CREDIT:K.SUTLIFF/SCIENCEHowCRISPR-Cas9

works?.Cas9技術流程KO：1weekcKO/KI：1-2monthsF0generation：1monthF1generation：3months無ES細胞限制，可選用小鼠品系多GLT率高，快速獲得雜合子（6months）.234651WildtypeAllele234651loxPloxP234651*ConditionalKOAllele(flox)Knock-inCas9cKO/KIstrategy.One-Stop

ServicesAnimalsdistributionBreedingservicesRederivation&cryopreservationVeterinaryservicesImport/exportTrainingCustomizedTG/KOMouseMousephenotypeResource

NanjingBiomedicalResearchInstitute

ofNanjingUniversity(NBRI)Research+.ThefirstcKOmouseinchina(2005)Thefirst

singleandmultiplegenesCas9KOmouseinchina(2013)ThefirstCas9cKOratinchina(2013)ThefirstKOmonkeyintheworld(2015)ThefirstKOdogintheworld

(2015)ProgressofNBRI’sCRISPR/Cas9Service.ModifiedfromSharpness&DePinho,Nat.Rev.DrugDiscovery,2006Abetterpre-clinicalevaluatingsystemisneededSuccessratebystagesofclinicaltestingforoncologycompounds.ChallengesinChemotherapyandtheDrugdevelopmentLowresponserateofSOCtodifferentpersons.Nature530,391(25February2016)doi:10.1038/nature.2016.19364“Retirement”ofNCI-60.Modifiedfrom/jax-mice-and-services1962ImmunodeficientMousestrains.NCGNOD-Prkdcem26Il2rgem26NjuNCG

micedevelopedbyNBRI(withindependentpropertyright),throughCRISPR/Cas9,donotexpressthe

Prkdc

genenortheX-linkedIl2rg

gene,arethemost

highlyimmunodeficientmice.Background：NOD/ShiLtJNjuPhenotype:

NOmaturelymphocytes,NONKcell，NOserumIg.

Readilysupportengraftmentofhumantransplantationstrategies..CommonnameNODscidgammaNODscidNudeMatureBcellsAbsentAbsentPresentMatureTcellsAbsentAbsentAbsentDendriticcellsDefectiveDefectivePresentMacrophagesDefectiveDefective

PresentNaturalkillercells

AbsentDefective

PresentComplementAbsentAbsent

PresentLeakinessVeryLowlowN/AIrradiationtoleranceLowLowHighLymphomaincidenceLowHighLowEngraftmentrateHighMediumLowMediansurvival>89wks36wks/ImmunodeficientMousestrains.NoThymusCharacteristicsofNCGmice.FACSdataofNCG.FlowCytometry-SpleenStrainsalymphocytes

NCGNOD-scidNODcontrolBcellsBcells(B220+)%0.231.4559.6TcellsCD4Tcells(CD3+CD4+)%5.844.1315.2CD8Tcells(CD3+CD8+)%0.190.797.1NKcellsNKcells(CD335+)%1.428.373.09FCM♀

miceat4wksDatafromNBRILifespan:NormalBreeding:NormalCharacteristicsofNCGmiceGrowthcurve.NCGNSGMutatedgenesPrkdc,Il2rgPrkdc,Il2rgBackgroundNOD/ShiLtJNju(Pure)NOD.B6(Congenic)GenehomozygosityVeryhighHighStrainvariationVerylowLowComparisonbetweenNCGandNSGmicePurebackgroundPhenotype&DataConsistency.CancerManagementandResearch2014:6431–436NSGmice

inCDX-Lungcancer.BT474MCF7MDA-MB-231NCGmice

inCDX-BreastcancerDatafromNBRI.NCG

inCDX-血癌細胞系接種量小于105DatafromNBRI.PDX(Patient-DerivedXenograft)modelModifiedfromNATUREREVIEWS/CLINICALONCOLOGYVOLUME9,JUNE2012.EstablishmentofPDXModels1.ImplantationsitesSubcutaneousRenalcapsuleOrthotopic:BrainIntestinalmammarygland...2.ValidationMorphologyGenomeGrowthCurve&ResponsetoSOC.PrimarytumorofPatientXenografts(P1)PrimarytumorofPatientXenografts(P1)Case1Case2DatafromNBRIEstablishmentofPDXModels.LeukemiaPDXPatientADatafromThird