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Knock-outs & Knock-ins

Knock-outs & Knock-ins. Mouse Knock-outs. Mouse Knock-ins. Mouse Knock-ins – WHY?. Test function of a specific residue or domain Create model for equivalent change in disease state Tag protein Test functional equivalence of genes and many, many more….

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Knock-outs & Knock-ins

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  1. Knock-outs & Knock-ins

  2. Mouse Knock-outs

  3. Mouse Knock-ins

  4. Mouse Knock-ins – WHY? • Test function of a specific residue or domain • Create model for equivalent change in disease state • Tag protein • Test functional equivalence of genes • and many, many more…

  5. Mouse Knock-ins – HOW?(Homologous recombination) • Two-step gene swap (+ & - selection) • Selection cassette very close to replacement region • May or may not be removed with Cre/lox

  6. Making a targeted mutation (“knockout”) in mouse cells Fig. 8-38 Griffiths, A., et al. (2002) Modern Genetic Analysis, 2ed.

  7. Two-step swap

  8. Selection cassette close to swap

  9. Fly knock-outs • Rationale: determine the function of particular genes in the genome • Strategy: use homologous recombination to insert mutated copy of the gene into the endogenous locus

  10. Alternative recombination strategies

  11. (d) The genetic cross scheme of the dArf6KO targeting experiment. Because dArf6 is on the second chromosome, a transgenic line carrying dArf6KO donor DNA (‘‘P{dArf6}Rpr1’’) on the third chromosome was used. w; Pin/CyO; Gal4221[w] stock was used to set up the screening cross in lieu of a regular Pin/CyO balancer stock. This allowed simultaneous selection against nonspecific targeting candidates while balancing the potential specific targeting candidates from the screening cross. P{dArf6}Rpr1*, linearized extrachromosomal dArf6KO donor DNA fragment. dArf6KO*, potential targeting events.

  12. Gene Knock-outs in Neurospora

  13. Knock-downs • Complete or incomplete reduction of gene function • Through heritable or non-heritable mechanisms that are not classical mutations

  14. Major Mechanisms for Knock-downs • RNAi (RNA interference)- aka. dsRNA, siRNA, (probably also antisense)- small double-stranded RNAs that interfere with normal gene activity • Morpholinos- synthetic oligonucleotide with typical base pairing properties

  15. Antisense RNA

  16. RNAi • Can induce transcriptional silencing • Can induce post-transcriptional degradation • Can induce translational repression

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