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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. FLP/FRT-Mediated Mitotic Recombination (Interchromosomal)

  4. What do we need? • Mutation of interest distal to the FRT • FRT near the centromere (preferably) • Source of FLP recombinase • Cell autonomous marker of genotype

  5. Employing cell markers for mitotic recombination

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

  7. 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

  8. 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

  9. Directed Mosaics

  10. Binary Expression System (GAL4/UAS)

  11. Directed Mosaics

  12. MARCM Scheme

  13. MARCM dominant marking system

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

  15. The “flp-out” cassette

  16. Flp-out clones with GFP marker

  17. Expression by flp-out cassette

  18. Mouse Mosaics: Cre/lox Recombination

  19. Mosaics used for Lineage Analysis

  20. Margolis and Spradling (1995). Development 121:3797-3807.

  21. The “Brainbow” System-Cell Tracing for the nervous system

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