MOSAIC ANALYSIS.

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Presentation transcript:

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 Determine “autonomy” of gene function Cell lineage analysis

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

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

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

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

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

Binary Expression System (GAL4/UAS)

Directed Mosaics

FLP/FRT-Mediated Mitotic Recombination (Intrachromosomal)

The “flp-out” cassette

Flp-out cassette to misexpress wingless

Binary Expression System (GAL4/UAS)

Flp-out clones with GFP marker

Expression by flp-out cassette

MARCM Scheme

MARCM dominant marking system

Mouse Mosaics: Cre/lox Recombination

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