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Knockout and transgenic mice: uses and abuses

Knockout and transgenic mice: uses and abuses. To create: homologous integration of DNA in embryonic stem cells Inserted DNA replaces normal gene at normal site on chromosome Usually homozygote for best expression Purpose: replace normal gene.

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Knockout and transgenic mice: uses and abuses

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  1. Knockout and transgenic mice: uses and abuses

  2. To create: homologous integration of DNA in embryonic stem cells Inserted DNA replaces normal gene at normal site on chromosome Usually homozygote for best expression Purpose: replace normal gene To create: injection of new DNA into fertilized egg Gene integrates randomly Multiple copies in tandem Expression level affected by integration site; may interrupt a gene Purpose: insert new genetic material Knockout mice Transgenic mice

  3. Basic steps—knockout • Pick a gene of interest • Knock it out or mutate it? • Create replacement construct • Inject into plasmid to cross over with gene of interest • Inject plasmid into stem cell and hope for recombination • Inject stem cell into blastocoel • Inject blastocyst into uterus

  4. Knockout variations Ablation of gene Homologous DNA DNA injected into plasmid Neo Exon 2 X X Plasmid with target gene Exon 1 Exon 2 Mutation of gene Desired mutation Homologous DNA DNA injected into plasmid Exon 1 Exon 2 X X Plasmid with target gene Exon 1 Exon 2

  5. Then you take your plasmid… And inject it into an ES cell Plasmid Neo Exon 2 X X Genomic DNA in ES cell Exon 1 Exon 2 Genomic DNA Neo Exon 2 which won’t produce a functional gene

  6. Creating knockout mice for fun and profit Injections to produce superovulation Two days after mating, harvest blastocysts and inject genetically targeted embryonic stem cells X X Inject blastocysts into uterus Pseudo-pregnant female Sterile male

  7. Chimeric males ES cells must enter their germline X 50% wild-type, 50% heterozygous (+/-) Breed hets 25% homozygous knockouts (-/-)

  8. Cool knockout tricks • Tissue specificity with Cre-LoxP system • Knock-ins: replacement of endogenous gene with a different one, for example CaMKII T305 animals, for constitutively active or inactive proteins

  9. Tissue-specific knockouts Cre-Lox system Cre recombinase snips out DNA between LoxP sites A tissue-specific promoter in front of Cre produces tissue-specific snipping Cre Transgenic mouse Tissue-specific promoter e.g. L7 (Purkinje) CaMKII (forebrain) Cre X Cre LoxP sites and everything in between them is excised Exon 1 Exon 2 Knockout only in promoter region Exon 2

  10. Basic steps—transgenic • Mutate or create a gene or fragment • Choose temporal regulation or not • Inject DNA construct into the male pronucleus of 1-cell embryos; hope for random insertion • Implant injected embryos into fallopian tubes

  11. Creating transgenics Injections to produce superovulation One day after mating, harvest 1-cell embryos and inject DNA construct X X Pseudo-pregnant female Sterile male Inject embryos

  12. 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 expression

  13. Cool transgenic tricks • Generalized overexpression • Reporter genes • Bicistronic reporters • Toxic genes • Dominant negatives • Targeted oncogenesis for immortalized tissue cultures • Tetracycline-regulated expression

  14. Reporter genes L7-GFP Purkinje cells glow green Use to identify Purkinje targets in brainstem Sekirnjak et al., 2003

  15. Bicistronic reporters IRES Promoter My Gene B-gal CAP-independent CAP-dependent IRES: internal ribosomal entry site Both genes are expressed from the same mRNA, so you can tell when and where your transgene has been expressed CAP is a sequence added in nucleus; normally it’s required for translation, but the IRES makes the second mRNA CAP-independent.

  16. Dominant negative transgenes Aim: to block protein kinase C (PKC) in Purkinje cells Problem: PKC has several isoforms, so knockouts aren’t effective Solution: PKCi transgene, which interferes with the regulatory portion of all PKCs, expressed under L7 promoter De Zeeuw et al., 1998

  17. 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

  18. Tetracycline regulation Aim: to avoid 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 can’t.

  19. So what’s the catch?

  20. So what’s the catch? • Difficult to knock out genes in certain chromosome regions, near centromere • Knockout animals are often homozygous lethal • Alternatively, KOs/Tgs may show no phenotype at all • Lack of temporal or spatial specificity may perturb development and other brain regions • Compensation by upregulation of other genes (e.g. PKC) • Transgenes can disrupt endogenous genes by landing in the middle of them

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