announcements exam i next tuesday (2/17) will cover all material through lecture this week
TRANSCRIPT
Announcements
• Exam I next Tuesday (2/17)• Will cover all material through lecture
this week
CONDITIONAL MUTATIONS
&MOSAIC ANALYSIS
CONDITIONAL MUTATIONS
• Permit temporal and/or tissue specific control over the loss of gene activity
Purposes of Conditional Mutations
• 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
Conditional Alleles
Small protein changes– Heat sensitive– Cold sensitive
Engineered domains– Drug-dependent
Recombination-induced genome modification (Mosaic analysis)
Isolation of conditional alleles
• Typically, generated at random– Point mutants– Labor intensive to identify– Rare, so not often done in multicellular
organisms• Can be created from known conditional
alleles in similar genes
Directed creation of ts alleles
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
Recombination-Induced Genome Modification
• Mediated by mitotic recombination• Interchromosomal• Intrachromosomal
• Must be induced (not normal)• Site-specific enzymes
» FLP recombinase» Cre recombinase
Mouse Mosaics: Cre/lox Recombination
There are various designs of targeting vector that can be used to generate loss- or gain-of-function alleles in ES cells.
©2008 by American Physiological Society
Adams D J , van der Weyden L Physiol. Genomics 2008;34:225-238
LOX FRT
Ligand-dependent Cre Recombinase
Tamoxifen-dependent Cre
Drosophila mosaics
Drosophila-FLP/FRT-Mediated Mitotic
Recombination (Interchromosomal)
SEM of Drosophila Compound Eye
w+
w-
FRT
FRT
FLP
FLP/FRT Targeted Mitotic Recombination
• Recombination Step
w+
w-
FRT
FRT
w+ / w-
red eyes
w+ / w-
red eyes
FLP/FRT Targeted Mitotic Recombination
• Segregation Option I: Outsides vs. Insides
• Segregation Option I: Outsides vs. Insides
w+
w-
FRT
FRT
w+ / w+
red eyes
w- / w-
white eyes
FLP/FRT Targeted Mitotic Recombination
• Segregation Option II: Tops vs. Bottoms
• Segregation Option II: Tops vs. Bottoms
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
Eye Differentiation in Drosophila
RTK Signal Transduction Pathway
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