Announcements Exam I next Tuesday (2/17) Will cover all material through lecture this week.

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Presentation 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: 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+ w-w- FRT FLP FLP/FRT Targeted Mitotic Recombination Recombination Step

w+w+ w-w- FRT w + / w - red eyes w + / w - red eyes FLP/FRT Targeted Mitotic Recombination Segregation Option I: Outsides vs. Insides

w+w+ w-w- FRT w + / w + red eyes w - / w - white eyes FLP/FRT Targeted Mitotic Recombination 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 FLP FLP/FRT Targeted Mitotic Recombination Recombination Step his-GFP

MEK + MEK - FRT MEK + /MEK - green cells MEK + /MEK - green cells FLP/FRT Targeted Mitotic Recombination Segregation Option I: Outsides vs. Insides his-GFP

MEK + MEK - FRT MEK + /MEK + green cells MEK - /MEK - white cells FLP/FRT Targeted Mitotic Recombination Segregation Option II: Tops vs. Bottoms his-GFP