announcements exam i next tuesday (2/17) will cover all material through lecture this week
TRANSCRIPT
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Announcements
• Exam I next Tuesday (2/17)• Will cover all material through lecture
this week
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CONDITIONAL MUTATIONS
&MOSAIC ANALYSIS
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CONDITIONAL MUTATIONS
• Permit temporal and/or tissue specific control over the loss of gene activity
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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
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Conditional Alleles
Small protein changes– Heat sensitive– Cold sensitive
Engineered domains– Drug-dependent
Recombination-induced genome modification (Mosaic analysis)
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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
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Directed creation of ts alleles
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MOSAIC ANALYSIS
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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
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Recombination-Induced Genome Modification
• Mediated by mitotic recombination• Interchromosomal• Intrachromosomal
• Must be induced (not normal)• Site-specific enzymes
» FLP recombinase» Cre recombinase
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Mouse Mosaics: Cre/lox Recombination
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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
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Ligand-dependent Cre Recombinase
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Tamoxifen-dependent Cre
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Drosophila mosaics
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Drosophila-FLP/FRT-Mediated Mitotic
Recombination (Interchromosomal)
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SEM of Drosophila Compound Eye
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w+
w-
FRT
FRT
FLP
FLP/FRT Targeted Mitotic Recombination
• Recombination Step
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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
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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
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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
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Employing cell markers for mitotic recombination
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Eye Differentiation in Drosophila
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RTK Signal Transduction Pathway
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MEK+
MEK-
FRT
FRT
FLP
FLP/FRT Targeted Mitotic Recombination
• Recombination Step
his-GFP
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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
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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