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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 1. 2. 3. 4. 5. 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 1. 2. 3. 4. In case of multiple alleles: complementation Rescue assays Morpholino Reverse genetics