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Research Techniques Made Simple: Generation of complete or tissue-specific knockout mice

Research Techniques Made Simple: Generation of complete or tissue-specific knockout mice. Lukas Scharfenberger, Tina Hennerici , Gábor Király , Sophie Kitzmüller , Marigje Vernoij , Julia G. Zielinski. What is a gene knockout?.

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Research Techniques Made Simple: Generation of complete or tissue-specific knockout mice

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  1. Research Techniques Made Simple:Generation of complete or tissue-specific knockout mice Lukas Scharfenberger, Tina Hennerici, GáborKirály, Sophie Kitzmüller, MarigjeVernoij, Julia G. Zielinski

  2. What is a gene knockout? • Manipulation of the DNA that results in the nonfunctionality or complete loss of the corresponding protein • Whole-body knockout: • knockout of a gene in every cell of the organism • Conditional knockout: • knockout of a gene in specific cell populations or organs

  3. Generation of gene-manipulated miceby gene targeting Isolation of embryonic stem (ES) cells Isolation of blastocyst in vitro cultivation of ES cells Transfection with targeting construct targeting construct 4 3 2 1 homologous recombination neo gene knockout neo neo 2 2 wildtype gene

  4. Generation of gene-manipulated miceby gene targeting Selection with neomycin Injection of ES cells into mouse embryo Breeding chimeric offspring Implantation into surrogate mother knockout mouse

  5. Conditional knockout Cre mouse floxed mouse created by gene targeting created by gene targeting neo 3 Cre 2 1 4 loxP loxP tissue-specific promoter crossing

  6. Conditional knockout Cre mouse Cre;;genefl/fl floxed mouse Cre neo 3 1 2 4 site-specific recombination loxP loxP degradation neo 2 1 4 3 Tissue-specific knockout

  7. Confirmation of gene knockout concept of Avcr1b knockout genotyping PCR Tissue-specific knockout in K14-Cre;;Avcr1bflox/flox mouse Qiu et al., 2011

  8. Overcome embryonic and early postnatal lethality of whole-body knockout mice • Tissue or organ autonomous analysis of a gene function • The combination of a tissue-specific expressing Cre mouse and floxed gene mouse offers a great variety of mouse models • Commercially available mouse lines save time and space Advantages of a conditional knockout model

  9. Limitations of a conditional knockout model • Not every human disease can be investigated by a mouse gene knockout • No temporal control of the gene knockout (inducible KO) • Some diseases have an underlying mechanism that results from overactivity of a gene expression (alternative: knockin model) • Redundancy of related gene products may compensate and thus not reveal the gene function

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