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• Determine “autonomy” of gene function

• Cell lineage analysis

FLP/FRT-Mediated Mitotic Recombination (Interchromosomal)

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

Employing cell markers for mitotic recombination

MEK+

MEK-

FRT

FRT

FLP

FLP/FRT Targeted Mitotic Recombination

•Recombination Step

his-GFP

MEK+

MEK-

FRT

FRT

MEK+/MEK-

green cells

MEK+/MEK-

green cells

FLP/FRT Targeted Mitotic Recombination

•Segregation Option I: Outsides vs. Insides

•Segregation Option I: Outsides vs. Insides

his-GFP

MEK+

MEK-

FRT

FRT

MEK+/MEK+

green cells

MEK-/MEK-

white cells

FLP/FRT Targeted Mitotic Recombination

•Segregation Option II: Tops vs. Bottoms

•Segregation Option II: Tops vs. Bottoms

his-GFP

Directed Mosaics

Binary Expression System (GAL4/UAS)

Directed Mosaics

MARCM Scheme

MARCM dominant marking system

FLP/FRT-Mediated Mitotic Recombination (Intrachromosomal)

The “flp-out” cassette

Flp-out clones with GFP marker

Expression by flp-out cassette

Mouse Mosaics: Cre/lox Recombination

Mosaics used for Lineage Analysis

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

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