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Mutagenesis and Genetic Screens Genome-Wide Phenotypic Analysis: “Phenomics” High-throughput genetic screens • Some genetic screens are relatively straightforward – e.g., For a visible phenotype like eye color • If phenotype is subtle or needs to be measured, the screen is more time consuming – Examples • Seed weight • Behavioral traits Industrial setting for screens 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. High-throughput genetic screen • Paradigm Genetics, Inc. performs “phenotypic profiling” • Take measurements of mutants’ physical and chemical parameters – e.g., plant height, leaf size, root density, and nutrient utilization • Different developmental times: 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. compare to wild type Phenomics Projects Generation of complete collection of mutants for entire genomes • Directed gene knock-outs for entire genome • Random insertion of transposons into all genes in genome • Random point mutations/indels in all genes in genome P element piggyBac Summary of P element Gene Disruption Project Potential for broad host range transposons in mutagenesis • Insertional mutagenesis (random) • Transgenic RNAi • Homologous recombination? (a la Drosophila) TILLING • Method for finding mutations produced by chemical mutagens in specific genes • Chemical mutagenesis – Usually produces point mutations – Very high mutagenic efficiency – Generally gives more subtle phenotypes than insertions • e.g., hypomorphs, temperature sensitive mutants 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 RTK Signal Transduction Pathway Mitotic Recombination • Must be induced (not normal) • DNA breaks (eg., X-rays) • Site-specific enzymes » FLP recombinase » Cre recombinase 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 FLP/FRT Targeted Mitotic Recombination •Recombination Step MEK+ his-GFP FRT FLP MEK- FRT FLP/FRT Targeted Mitotic Recombination •Segregation Option I: Outsides vs. Insides MEK+/MEKgreen cells MEK+ his-GFP FRT •Segregation Option I: Outsides vs. Insides MEK- FRT MEK+/MEKgreen cells FLP/FRT Targeted Mitotic Recombination •Segregation Option II: Tops vs. Bottoms MEK+/MEK+ green cells MEK+ his-GFP FRT •Segregation Option II: Tops vs. Bottoms MEK- FRT MEK-/MEKwhite cells Binary Expression System (GAL4/UAS) Directed Mosaics MARCM Scheme MARCM dominant marking system