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MOSAIC ANALYSIS

MOSAIC ANALYSIS. MOSAIC ANALYSIS - WHY? Purposes of Mosaic Analysis. Examine later and/or tissue-specific functions of a gene required for viability Bypass lethality to examine later function Determine where gene function is required Find which tissue is the source of gene activity

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MOSAIC ANALYSIS

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  1. MOSAIC ANALYSIS

  2. MOSAIC ANALYSIS - WHY?Purposes of Mosaic Analysis • Examine later and/or tissue-specific functions of a gene required for viability • Bypass lethality to examine later function • Determine where gene function is required • Find which tissue is the source of gene activity • Determine “autonomy” of gene function • Cell lineage analysis

  3. MOSAIC ANALYSIS – HOW?General Strategies for Mosaic Analysis • Nuclear or Cellular Transplantation • Mitotic Chromosomal Loss • Mitotic Recombination • Interchromosomal • Intrachromosomal

  4. Mitotic Recombination • Must be induced (not normal) • DNA breaks (eg., X-rays) • Site-specific enzymes • FLP recombinase • Cre recombinase

  5. FLP/FRT Targeted Mitotic Recombination • Recombination Step MEK+ his-GFP FRT FLP MEK- FRT

  6. FLP/FRT Targeted Mitotic Recombination • Segregation Option I: Outsides vs. Insides MEK+/MEK- green cells MEK+ his-GFP FRT • Segregation Option I: Outsides vs. Insides MEK- FRT MEK+/MEK- green cells

  7. FLP/FRT Targeted Mitotic Recombination • Segregation Option II: Tops vs. Bottoms MEK+/MEK+ green cells MEK+ his-GFP FRT • Segregation Option II: Tops vs. Bottoms MEK- FRT MEK-/MEK- white cells

  8. Binary Expression System (GAL4/UAS)

  9. Directed Mosaics

  10. FLP/FRT-Mediated Mitotic Recombination (Intrachromosomal)

  11. The “flp-out” cassette

  12. Flp-out cassette to misexpress wingless

  13. Binary Expression System (GAL4/UAS)

  14. Flp-out clones with GFP marker

  15. Expression by flp-out cassette

  16. MARCM Scheme

  17. MARCM dominant marking system

  18. Mouse Mosaics: Cre/lox Recombination

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