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Forward Genetic Screens:Strategies and challenges
HarwinGoFish
22 July 2015
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
Things to consider
1. Phenotypes to screen for2. Methods of mutagenesis3. Identification of mutagenized gene4. Degree of saturation5. Proof of candidate gene
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
2. Methods of mutagenesis
Gamma‐ray irradiation
Chemical mutagenesis
Insertional mutagenesis
Gamma‐ray irradiation
Kimmel, 1989
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
Chemical mutagenesis
Lawson and Wolfe, 2011
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
Insertional mutagenesis
Lawson and Wolfe, 2011
Insertional mutagenesis
Amsterdam et al., 1999
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)
Summary: mutagenesis methods
Amsterdam and Hopkins, 2006
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
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)
Positional cloning
Zhou and Zon, 2011
• 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
Mutagenesis + Whole genome‐seq
Zebrafish Mutation Project (ZMP)
Problems with ZMP
1. Low depth of sequence‐ Sequencing gametes is less sensitive
2. Difficulty in recovering found mutations, husbandry
3. Space constraints
Positional cloning + Seq: A better approach?
Obholzer et al., 2012
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
5. Proof of Candidate Genes
1. In case of multiple alleles: complementation2. Rescue assays3. Morpholino4. Reverse genetics