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Mutagenesis and Genetic
Screens
Genome-Wide
Phenotypic Analysis:
“Phenomics”
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
TILLING in Arabidopsis I
• EMS used to
mutagenize
Arabidopsis
• Grow individual
mutagenized lines
• Make primers flanking
gene of interest
• Amplify using PCR
EMS
mutagenize
seed
gene Z
WT
gene Z
mutant
PCR amplification
from wild type
and mutant
WT
mutant
TILLING in Arabidopsis II
• Denature DNA from
pools of mutant lines
• Allow to hybridize to
wild-type DNA
• Detect mismatches in
hybridized DNA
– Denaturing HPLC
– Cel I enzyme cuts at
mismatches
• Sequence to identify
site of mutation
ATGCGGACTG
TACGCCGGAC
ATGCGG
CTG
TACGCC
GAC
Cel 1
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
SEM of Drosophila Compound Eye
Eye Differentiation in
Drosophila
RTK Signal Transduction Pathway
MOSAIC ANALYSIS – HOW?
General Strategies for Mosaic Analysis
• Nuclear or Cellular Transplantation
• Mitotic Chromosomal Loss
• Mitotic Recombination
• Interchromosomal
• Intrachromosomal
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)
FLP/FRT Targeted Mitotic Recombination
•Recombination Step
w+
FRT
FLP
w-
FRT
FLP/FRT Targeted Mitotic Recombination
•Segregation Option I: Outsides vs. Insides
w+ / wred eyes
w+
FRT
•Segregation Option I: Outsides vs. Insides
w-
FRT
w+ / wred eyes
FLP/FRT Targeted Mitotic Recombination
•Segregation Option II: Tops vs. Bottoms
w+ / w+
red eyes
w+
FRT
•Segregation Option II: Tops vs. Bottoms
w-
FRT
w- / wwhite eyes
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