实验技术课小鼠基因操作2013

1、Genetic mouse ms forbiological research袁Current genetic manipulation systems used forgene expression or deletion studiesMaking a transgenic mouse to express a gene ofinterest to see its effect on tissue or cell development and functionKnock-out a gene to see its effect on tissue orcell development a

2、nd functionGetting to a PhenotypePhenotypic AnalysisTransgenic mCIB1 construct mapBamH IXhoXhoBamH IPGKMouse CIB1 + c-mycPolyA Tail1.6kb0.68kb0.35kbProbe 2Probe 1Probe CBamH: 2.6kbTransgenic CIB1 mouse express mCIB1- myc mRNAMouse 2 CIB1-myc vectorMouse 1Mouse 2Mouse 3Primers inside mCIB15 Primer in

3、 mCIB1 3 Primer in Myc onlyMyc tagged CIB1 protein is not overexpressed in transgenic miceCIB1-mycCIB1WTTGTGWTTGNo phenotype was found in this transgenic micePitfalls for making transgenic miceThe transgenic mice do not express your proteinThe transgenic mice do not overexpress yourproteinThe transg

4、enic mice overexpress your protein butthe phenotype is marginal and you cannot make aWhy are these pitfallsYour construct has faultYou insert your transgene into wrong places(bad luck)You did everything right and the transgenicmice express but not overexpress your proteinCurrent genetic manipulation

5、 systems used forgene expression or deletion studiesMaking a transgenic mouse to express a gene ofinterest to see its effect on tissue or cell development and functionKnock-out a gene to see its effect on tissue or celldevelopment and functionTraditional knockout studiesGene of interestExon 1Exon 2E

6、xon 3Exon 4Targeted allelesExon 1 ReporterNeo Exon 3 Exon 4Exon 1NeoExon 3Exon 4Delete a mouse gene in an old fashion wayScreening the BAC clone that containing CIB1Cloning tSequencing tomic DNA that franking the CIB1omic DNAMaking the knockout constructScreening targeted ES cellsBlastocyst injectio

7、n with right ES cloneGet your finger crossedA classic gene targeting vector for conventional knockout construct5 Pac-Bam-Sal-Hpa-Kpn-Hind-Xba-3(Long arm)Not I (linearization)Xho I Sfi I Pml I Bcl I Bbs I 5 Nhe I(Short arm)osdupdel6142 bpPGK TKOliver SmithiesNobel LaureateKnockout strategy for mCIB11

8、5.0kbBamH IHXPBcBamH IWild-type allele0.55kb deletion3 primerNBNeo-primer3 homology5 homologyTargeting vector4.5kbNeo 1.2kb1.2kbTK3.0 kbBamH IBamH IBamH IMutant alleleSouthern Probe123671234567Verification of Cib1-/- mice by Southern blot & PCR+/+/-/-+/+/-/-/-15 kb8 kbKO primersWT primers3 kbDig

9、estion:BamH ICib1 null mice fertility assessmentMaleGenotype strainFemaleGenotypestrainMatingpairsLittersper pairAverageLitter size9.5 ±9.3 ±9.3 ±+/+C57BL/6+/+/-/-C57BL/6mixed mixed6666661.41.21.98.7 ±9.0 ±+/-mixed+/+/-/-C57BL/6mixedmixed6666661.21.410.2 ± 2.5-/-mixed+/

10、+/-/-C57BL/6mixed mixed666000NANA NANote: Mice on mixed background were derived from offspring of strains C57BL/6 and 129J. Fertility assessment was stopped when total litters reach six for a particular breeding pair.What you can do if you want to observe tissue-specific function of the protein of i

11、nterestGene targeting strategies with LoxP sitesGene of interestExon 1Exon 2Exon 3Exon 4Targeted alleleDifferent alleleExon 1 ReporterNeoExon 2 Exon 3 Exon 4Exon 1Exon 2Exon 3Exon 4Lox Sites8 bp spacer13 bp Cre binding13 bp Cre binding ATAACTTCGTATA GCATACAT TATACGAAGTTAT LoxPLoxP sequence consists

12、of an asymmetric 8 bp sequence in between with two sets of flanking palindromic 13 bp sequences.Knockout strategies with LoxP sitesGene of interestExon 1Exon 2Exon 3Exon 4Targeted alleleDifferent alleleExonReporterNeoExonExonExonExon 1Exon 2Exon 3Exon 4Cre-mice (promoter-driventissue specific expres

13、sion)Desired mutation bearing miceHow to make a knockout mouse inpost-genomic daysGenomic DNA comparison of mouse Nephrocan with other species(Gene position: chr10: 52,111,414-52,124,410, UCSC Genome Browser Program )Up and down 2kb:chr10, position 52,106,778-52,127,413Up and down till next ORF: chr

14、10, position 52,100,778-52,138,413Bac clones covering Nepn from C57BL/6 and other libraries()Nepn (Ch10)Start:52111414Stop:52124410Clone OrientationCloneorderedClonesStart (bp)Stop (bp)Length (bp)Clone WeightEnd1End2MSMG01-91M7RP23-236F22 RP23-87E19 MSMG01-522O17 RP23-383C2RP23-412B21MSMG01-94J19 RP

15、23-167J16520196365210071152100732520231085203857351943313520196425206910652118077522825805228696852196638522384305213003352118077522644529844118186918623617353019985718672098435195346HighestHighest Highest Highest Highest Highest HighestHighest+-YesYesBAC clones covering Nepn from 129S7/AB2.2 librar

16、y()Clone of 129S7StartStopClone orderedbMQ-236D155206673852200356133,618YesbMQ-369M215200557252110754105,182bMQ-365P205204320252160078123,876Nepn (Ch10)521114145212441012997BAC recombineering for generating LCA targeting vectorF repliconBAC clone Area of interestpLCA.71/2272.NTK1F repliconBAC cloneM

17、CS DTpBS.DT-A2HA1HA2 DTGene targeting vectorpuDtkEM7neopuDtkEM7neopuDtkEM7neoMCS: multicloning sites HA: homology armLox71 siteLox66 site Lox 2272 siteFRT siteCan we freely exchange contentsbetween loxP sites?Targeted alleleExon 1Exon 2Exon 3Exon 4loxPiloxPLoxed cassetteacceptor alleleloxPiloxPExcha

18、nge vectorCreCassetteexchange alleleFLPeNew alleleNewNewHygroRNewHygroRNeoR HSC-tKloxP-inverted loxP StrategyLox Sites8 bp spacer13 bp Cre binding13 bp Cre binding ATAACTTCGTATA GCATACAT TATACGAAGTTAT ATAACTTCGTATA GGATACTT TATACGAAGTTAT TACCGTTCGTATA GCATACAT TATACGAAGTTAT ATAACTTCGTATA GCATACAT TA

19、TACGAACGGTA TACCGTTCGTATA GCATACAT TATACGAAGTTAT LoxPLox2272Lox71Lox66Lox66/71Efficient recombinationloxP + loxP lox2272 + lox2272 lox71 + lox66Inefficient recombinationloxP + lox2272 lox71/66 + loxPRecombinase-mediated cassette exchange (RMCE)lox71lox2272Acceptorallelelox66lox2272NewHygroRExchangev

20、ectorCreCassetteexchange alleleFLPeNew alleleNewNewHygroRpuDtkAdvantage of RMCEThe design and assembly of exchange vectors is simpler than that for gene targeting vectors.The design can easily be incorporated a negative selection strategy to aid identification of RMCE events.High efficiency in recom

21、bineering events when compared with homologous recombination in ES cells.It a targeted insertion rather than random integration so transgene can be put in at desired place.RMCE strategypUDTK-EM7neoRosa26 LCALox71Lox2272Exon 2ExchangeVectorLox66PromoterReporter HygroLox2272CreRosa26-RLox71/66Promoter

22、Reporter HygroLox2272Exon 2Advantage over conventional methodsCan quickly generate new ES cells for analysis with RMCE technologyCan analyze gene expression in an in vitro culture systemCan efficiently generate mice with using such constructs if in vitro analysis warranted such need Put big enough p

23、romoter region into the LCA ofRosa26 willally reproduce the true in vivoexpression of gene of interestAvoid possible haplo-insufficiencyMouse Rosa26 locus (to overcome the pitfallof generating transgenic mice)No expression of its own geneGene knocked-in at this locus seemed can be ubiquitously expre

24、ssed without been down- regulated (often seen in random targeting of transgene)Has been used successfully to generate mouse reporter linesHuman Rosa26 has been identified and experimented with similar studies successfullyMouse Rosa26 structure and its LCA structure50001000015000200002500030000350004

25、0000Rosa26wtBstBINsiINsiIBstBI12P1P2Targeting VectorpUDTK-EM7neoDT-AHomologous RecombinationRosa26BstBIBstBINsiINsiIBstBItm12Lox71,lox2272 sitesAlleles: Wild type =Rosa26wtLoxed cassette acceptor =Rosa26tm1Various RMCE constructs1.Basic-CFPCFPNeoglobin splice pA 2.CFP-BG-pA3.CFP-SV40pA SV40 polyA 4.

26、Basic-CFP-EN-globin splice pANeo5.Basic-Cherryglobin splice pA6.CFP-Ires-PuroRNeo-globin splice pACFP.Ires.Puro-CFP-globin polyA-Rosa 5 elementNeoCherryRosa 5 elementRosa 5 elementNeoCFPRosa 5 elementNeoCFPRosa 5 elementRosa 5 element小鼠体内干细胞多色萤光追踪模型Making transgenic mice to expressed protein ofinter

27、est at specific locusNephrocan expresses during pancreatic developmentNephrocan (Nepn) is a novelmember of the small leucine rich repeat protein family. It was first identified as a protein expressed during kidney development and in adult kidneys.Nephrocan was recently found tobe expressed specifica

28、lly at the definitive endoderm stage (E7.0- 9.5) during pancreatic development in mice by two papers using microarray analysis(Sherwood., 2007) andSAGE methods (Hou., 2007).A,B,D: Lateral and posterior viewF: Lateral and ventral viewGenomic Location of Mouse NephrocanChromosome location:GenBank ID:

29、6665010 B335GopcNepnNus144,994 bp9,533 bp12,996 bp12,955 bp19,206 bpPut in fluorescent markerin a different wayE1E2E3HA1HA3 HA4HA2BAC.NepnpL451-Cherry hasbeen madeATGpL451-CherryCherry reporterhere has beeninserted into BAC.Nepn HA4 HA3 Cherry.NLSIRESPuroRPolyANeoRATGBAC.Nepn.Cherry.NeoCherry.NLSIRE

30、SPuroRPolyANeoR HA4 HA3HA4PuroRPolyANeoRCherryIRESATGBAC.Nepn.Cherry.NeoHA1HA2pBR322V.66.2272.HA1.HA2Retrieval VectorE1 E2 E3CherryIRESPuroRPolyANeoRATGpBR322V.Nepn.CherryExchange VectorBamH1BamH1HygroLox2272Lox66 Nepn DNAFRTHA3Overview of pancreas developmentOct4Foxa2Hnf1bHnf6Nkx2.2InsNanogSox2 ECA

31、DCXCR4Sox17Hnf4aHlxb9Ptf1a Pdx1NKx6.1Pax4 Ngn3GCGGHRLMafANepnWhat you can do if you want to observe time-specific function of the protein of interestEvidence of transdifferentiation by overexpressionof transcription factorsOct4Foxa2CXCR4Sox17NepnHnf1bHnf4aHnf6Hlxb9 Ptf1a Pdx1Nkx2.2NKx6.1Pax4Ngn3InsG

32、CG GHRLMafANanogSox2ECADThe forced over-expression of Pdx1, Ngn3 and MafA in pancreatic acinarcells causes them to change into insulin producing beta cells.Zhou., Nature 2008Exchange vectors made for rtTA micePtf1artTArtTA-globin splice & pAPdx-1rtTArtTAHygroPdx1-globin splice & pALox 2272 s

33、iteloxP sitetranslationalenhancerFRT siteLox66 siteHygroPtf1aReverse tetracycline transactivator systemin mice(Often called Tet-On)rtTAMouse 1OffrtTAOn- Dox+DoxMouse 2Factor 2XFactor 1Factor 1Factor 2Factor 2TetOTetOFactor 1rtetRVP16Potential mouse lines for TRE systempromotergenelocusgenesXRosa26AMafA/mCherryPdx1rtTARosa26ANgn3/CFPPtf1artTAPdx1Pdx1/eGFPMice that express genes under controlof a TREMice that express rtTA under Ptf1a orPdx1 controlA few frequently used cre mice forblood cell studyTie2-CreVav1-CreVE-Cadherin-Cre ER-CreMX1-Cre建内中胚层干细胞示踪技术体系Va