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1、Knockout and transgenic mice:uses and abuses,Knockout mice Transgenic mice,To create:homologous integration of DNA in embryonic stem cellsInserted DNA replaces normal gene at normal site on chromosomeUsually homozygote for best expressionPurpose:replace normal gene,To create:injection of new DNA int
2、o fertilized eggGene integrates randomlyMultiple copies in tandemExpression level affected by integration site;may interrupt a genePurpose:insert new genetic material,Basic stepsknockout,Pick a gene of interestKnock it out or mutate it?Create replacement constructInject into plasmid to cross over wi
3、th gene of interestInject plasmid into stem cell and hope for recombinationInject stem cell into blastocoelInject blastocyst into uterus,Knockout variations,Ablation of gene,Neo,Exon 2,Exon 1,Exon 2,X,Homologous DNA,X,Plasmid with target gene,DNA injected into plasmid,Exon 1,Exon 2,Exon 1,Exon 2,Mut
4、ation of gene,Desired mutation,Plasmid with target gene,DNA injected into plasmid,X,X,Homologous DNA,Then you take your plasmid,Neo,Exon 2,And inject it into an ES cell,Exon 1,Exon 2,X,X,Plasmid,Genomic DNA in ES cell,Neo,Exon 2,Genomic DNA,which wont produce a functional gene,Creating knockout mice
5、 for fun and profit,Injections to produce superovulation,X,X,Sterile male,Pseudo-pregnant female,Two days after mating,harvest blastocysts and inject genetically targeted embryonic stem cells,Inject blastocysts into uterus,Chimeric malesES cells must enter their germline,X,50%wild-type,50%heterozygo
6、us(+/-),Breed hets,25%homozygous knockouts(-/-),Cool knockout tricks,Tissue specificity with Cre-LoxP systemKnock-ins:replacement of endogenous gene with a different one,for example CaMKII T305 animals,for constitutively active or inactive proteins,Tissue-specific knockouts,Cre-Lox system Cre recomb
7、inase snips out DNA between LoxP sites A tissue-specific promoter in front of Cre produces tissue-specific snipping,Tissue-specific promoter,Cre,Transgenic mouse,e.g.L7(Purkinje)CaMKII(forebrain),Exon 1,Exon 2,X,Exon 2,LoxP sites and everything in between them is excised,Cre,Knockout only in promote
8、r region,Cre,Basic stepstransgenic,Mutate or create a gene or fragmentChoose temporal regulation or notInject DNA construct into the male pronucleus of 1-cell embryos;hope for random insertionImplant injected embryos into fallopian tubes,Injections to produce superovulation,X,X,Sterile male,Pseudo-p
9、regnant female,One day after mating,harvest 1-cell embryos and inject DNA construct,Creating transgenics,Inject embryos,Many offspring will carry the inserted DNA;only some will express it usefully,X,Several transgenic lines stemming from different F1,Breed selectively,Usually both+/+and+/-show expr
10、ession,Cool transgenic tricks,Generalized overexpressionReporter genesBicistronic reportersToxic genesDominant negativesTargeted oncogenesis for immortalized tissue culturesTetracycline-regulated expression,Reporter genes,L7-GFPPurkinje cellsglow greenUse to identifyPurkinje targetsin brainstem,Seki
11、rnjak et al.,2003,Bicistronic reporters,My Gene,B-gal,IRES,IRES:internal ribosomal entry siteBoth genes are expressed from the same mRNA,so you can tell when and where your transgene has been expressedCAP is a sequence added in nucleus;normally its required for translation,but the IRES makes the sec
12、ond mRNA CAP-independent.,Promoter,CAP-independent,CAP-dependent,Dominant negative transgenes,Aim:to block protein kinase C(PKC)in Purkinje cells Problem:PKC has several isoforms,so knockouts arent effective Solution:PKCi transgene,which interferes with the regulatory portion of all PKCs,expressed u
13、nder L7 promoter,De Zeeuw et al.,1998,Dominant negative transgenes,Aim:to block BDNF signalling through TrkB Problem:BDNF can activate another receptor as well(p75)Solution:TrkB-Tc transgene,which allows BDNF binding but prevents signalling,Saarelainen et al.,2003,Tetracycline regulation,Aim:to avoi
14、d developmental effects of transgene expression Solution:Tet system,where a transgenic producing tTA is crossed with a transgenic with the tet-O promoter.tTA normally permits tet-O transcription,but in the presence of doxycycline it cant.,So whats the catch?,Difficult to knock out genes in certain c
15、hromosome regions,near centromere Knockout animals are often homozygous lethalAlternatively,KOs/Tgs may show no phenotype at allLack of temporal or spatial specificity may perturb development and other brain regionsCompensation by upregulation of other genes(e.g.PKC)Transgenes can disrupt endogenous genes by landing in the middle of them,So whats the catch?,
