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Round table discussion: Genetic Screens and Mutagenesis Methods 1) Mutagenesis
• Chemical • Radiation • Retroviruses and transposon insertional vectors • Specific gene targeting: TALENs, CRISPR, Zinc finger nucleases • The future: homologous recombination
2) Genetic Screens Forward: standard 3 generation screening, haploids, gynogenetic diploids, maternal-effect and adult screens
Reverse: Mutation detection approaches: resequencing, TILLING, retroviral insertions, TALENs, CRISPRs, zinc finger nucleases
γ-rays • Deletions (usually large) & translocations (often not mendelian) • Mutation rate: 1/200 F1 individuals • Utility: null phenotype, removing adjacent duplicate genes,
non-complementation screen.
ENU (ethylnitrosourea) • spermatogonial treatment yields point mutations
---Mutation rate: 1/1,000 F1 individuals ---Utility: nulls and hypomorphs, unbiased wrt genes mutated
• sperm treatment yields points and deletions Mutation rate: 1/200
Mutagens
!
Leal et al., 2009; Schulz et al., 2009
Zebrafish Spermatogenesis
ENU induces mutations at all stages of spermatogenesis. Clones of mutations can occur, so keep track of F1s from each Founder male.
Spermatogonial stem cell
γ-rays • Deletions (usually large) & translocations (often not mendelian) • Mutation rate: 1/200 F1 individuals • Utility: null phenotype, removing adjacent duplicate genes,
non-complementation screen.
Insertional mutagenesis • retroviral (MLV-VSV) insertion into or close to gene (N. Hopkins) • Mutation rate: 1/10,000 F1 individuals (some hotspot genes) • Transposable element insertions (Tol 2)—gene traps • Rapid cloning!
ENU (ethylnitrosourea) • spermatogonial treatment yields point mutations
---Mutation rate: 1/1,000 F1 individuals ---Utility: nulls and hypomorphs, unbiased wrt genes mutated
• sperm treatment yields points and deletions Mutation rate: 1/200
Mutagens
Standard 3 generation screen Haploids Gynogenetic diploids
Pros Cons
All stages and genomic regions accessible Mendelian ratios Natural breeding is the only “technique”
Requires only 1 generation All genomic regions accessible Mendelian ratios
Requires only 1 generation Diploids screened: all stages accessible
Biased against telomeric regions Non-Mendelian ratios EP procedure reduces throughput Background of EP effects
Useful only at early stages Background effects may obscure some phenotypes
Requires two generations Labor-intensive More tanks required
Zebrafish Zygotic Mutant Screen--Natural Crosses G0
F1
F2
F3
Screen F3 embryos morphologically at 1 dpf,
2 dpf, 5 dpf.
*/+ +/+
50% */+ 50% +/+
F1
F2
Early pressure (diploid)!
Block 2nd polar body formation with ‘Early Pressure’ (EP)!
Gynogenetic diploid!
F1
F2
2/3 of all mutants comprise 3 general phenotypic classes
Widespread cell death
Heart edema/poor circulation
Widespread cell death starting in CNS
General retardation in development
Wild type
Wild type
What types of genes might be mutated?
General “housekeeping” genes. What are they?
Web Resources for acquiring mutants without doing a screen: ZIRC (http://zebrafish.org/zirc/home/guide.php) ZFIN (http://zfin.org/) ZMP (http://www.sanger.ac.uk/Projects/D_rerio/zmp/) also TILLING Requests (https://webapps.fhcrc.org/science/tilling/index.php)