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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