GENOME SEQUENCE ANALYSIS OF DNA-A FROM OKRA YELLOW VEIN MOSAIC VIRUS ISOLATES
MOSAIC ANALYSIS
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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