Download Forward Genetic Screens: Strategies and challenges

Forward Genetic Screens:
Strategies and challenges
Harwin
GoFish
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
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Phenotypes to screen for
Methods of mutagenesis
Identification of mutagenized gene
Degree of saturation
Proof of candidate gene
1. Phenotypes to screen for
1. Morphology, lethality
2. Maternal phenotypes
3. Specific embryonic phenotype (Ab staining, ISH, nervous and hematopoietic systems)
4. Modifier screens
5. Temperature sensitive screen
6. Regulatory elements
2. Methods of mutagenesis
Gamma‐ray irradiation
Insertional mutagenesis
Chemical mutagenesis
Gamma‐ray irradiation
Kimmel, 1989
Gamma‐ray irradiation
Time: Mutagenized sperm or embryos at mid‐blastula stage
Effect: Large deletions / translocations
Pros:
Relatively fast
High mutagenic rate (~1.2 hit per 100,000 genome/rad, higher with increasing rad)
Cons:
High lethality (non‐specific)
Hard to map/maintain lines
Chemical mutagenesis
Lawson and Wolfe, 2011
Chemical mutagenesis
Time: Premeiotic sperm
Effect: Point mutations
Pros:
Fast mutagenesis and family generation
Highest mutagenic rate (3 hits/gene/1000 genomes screened)
More random than insertional mutagenesis
Cons:
Lots of silent missense mutations
Positional cloning takes FOREVER
Need multiple outcrosses to divergent background for mapping
Insertional mutagenesis
Lawson and Wolfe, 2011
Insertional mutagenesis
Amsterdam et al., 1999
Insertional mutagenesis
Time: Midblastula embryos
Effect: Large insertion and gene silencing
Pros:
Positional cloning is super easy
Every integration results in silencing
Cons:
Mutagenesis rate is lower than ENU
Mutagenesis is very labor intensive
Slight 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
Amsterdam and Hopkins, 2006
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
3. Identification of mutagenized gene
Positional Cloning
mutation
Chromosomes with mutation
= Simple Sequence Length Polymorphisms (SSLPs)
‐ Polymorphic markers close to mutation will segregate with mutation more frequently than markers further away
Positional cloning
Zhou and Zon, 2011
Positional cloning
• 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!
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 Saturation
A. How efficient is the mutagenesis?
B. How many hits on the same gene? Also depends on the gene size
Mullins et al., 1994
5. Proof of Candidate Genes
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In case of multiple alleles: complementation
Rescue assays
Morpholino
Reverse genetics