Forward Genetic Screens: Strategies challenges › ... › gofish_forward_genetic_screen.… ·...
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Forward Genetic Screens:Strategies and challenges
HarwinGoFish
22 July 2015
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Forward genetics
Phenotype Gene
Several advantages:‐ Starting point is a strong phenotype
‐ Unbiased approach possibility to find new regulators of certain process
‐ Able to obtain large number of genes involved in the same process
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Things to consider
1. Phenotypes to screen for2. Methods of mutagenesis3. Identification of mutagenized gene4. Degree of saturation5. Proof of candidate gene
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1. Phenotypes to screen for
1. Morphology, lethality2. Maternal phenotypes3. Specific embryonic phenotype (Ab staining,
ISH, nervous and hematopoietic systems)4. Modifier screens5. Temperature sensitive screen6. Regulatory elements
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2. Methods of mutagenesis
Gamma‐ray irradiation
Chemical mutagenesis
Insertional mutagenesis
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Gamma‐ray irradiation
Kimmel, 1989
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Time: Mutagenized sperm or embryos at mid‐blastula stage
Effect: Large deletions / translocations
Pros:Relatively fastHigh mutagenic rate (~1.2 hit per 100,000 genome/rad, higher with increasing rad)
Cons:High lethality (non‐specific)Hard to map/maintain lines
Gamma‐ray irradiation
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Chemical mutagenesis
Lawson and Wolfe, 2011
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Chemical mutagenesisTime: Premeiotic sperm
Effect: Point mutations
Pros:Fast mutagenesis and family generationHighest mutagenic rate (3 hits/gene/1000 genomes screened)More random than insertional mutagenesis
Cons:Lots of silent missense mutationsPositional cloning takes FOREVERNeed multiple outcrosses to divergent background for mapping
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Insertional mutagenesis
Lawson and Wolfe, 2011
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Insertional mutagenesis
Amsterdam et al., 1999
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Insertional mutagenesisTime: Midblastula embryos
Effect: Large insertion and gene silencing
Pros:Positional cloning is super easyEvery integration results in silencing
Cons:Mutagenesis rate is lower than ENUMutagenesis is very labor intensiveSlight bias towards open regions of the genome (higher insertion rate at 5’ ends)
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Summary: mutagenesis methods
Amsterdam and Hopkins, 2006
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3. Identification of mutagenized gene
Insertional mutagenesis has the upperhand!
Inverse PCR + BLASTing known sequence = rapid mapping!
Some technical problems with highly similar regions in the past, but with better genome sequence this is minimized
Amsterdam and Hopkins, 2006
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Positional Cloning
3. Identification of mutagenized gene
Chromosomes with mutation
mutation
‐ Polymorphic markers close to mutation will segregate with mutation more frequently than markers further away
= Simple Sequence Length Polymorphisms (SSLPs)
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Positional cloning
Zhou and Zon, 2011
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• Labor intensive• Need ~2000 mutants to be able to map to 0.1cM
• With better genome, still needs ~400 mutants to map to 1cM, and sequence genes in between
• There are sites with minimal recombination in the genome!
Positional cloning
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Mutagenesis + Whole genome‐seq
Zebrafish Mutation Project (ZMP)
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Problems with ZMP
1. Low depth of sequence‐ Sequencing gametes is less sensitive
2. Difficulty in recovering found mutations, husbandry
3. Space constraints
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Positional cloning + Seq: A better approach?
Obholzer et al., 2012
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4. Degree of SaturationA. How efficient is the mutagenesis?
Mullins et al., 1994B. How many hits on the same gene?
Also depends on the gene size
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5. Proof of Candidate Genes
1. In case of multiple alleles: complementation2. Rescue assays3. Morpholino4. Reverse genetics