Presentation on theme: "Round table discussion: Genetic Screens and Mutagenesis Methods"— Presentation transcript:
1Round table discussion: Genetic Screens and Mutagenesis Methods • Chemical• Radiation• Retroviruses and transposon insertional vectors• Zinc finger nucleases• The future: homologous recombination?2) Genetic ScreensForward: standard 3 generation screening, haploids, gynogenetic diploids, maternal-effect and adult screensReverse: Mutation detection approaches: resequencing, TILLing,retroviral insertions, zinc finger nucleases, TALENsADD spermatogenesis stage to explain mutating sperm v various spermatogonial stages
2Mutagensg-raysDeletions (usually large) & translocations (often not mendelian)Mutation rate: 1/200 F1 individualsUtility: 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 mutatedsperm treatment yields points and deletionsMutation rate: 1/200Insertional mutagenesisretroviral (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 trapsRapid cloning!
3+/-(difficult, expensive) Relative Attributes of Genetic SystemsYeast Drosophila C. elegans zebrafish mouseForward+++++++++/-(difficult, expensive)(phenotype-based)++Reverse+++++(gene-based)homologousrecombinationP-elementsRNAi*homologous recomb.RNAi*Mutation DetectionMorpholinos*Mutation DetectionZFN, TALENshomologousrecombination* Not really genetics
4Cons Pros Standard 3 generation screen Haploids Gynogenetic diploids All stages and genomic regions accessibleMendelian ratiosNatural breeding is the only“technique”Standard 3 generationscreenHaploidsGynogenetic diploidsRequires two generationsLabor-intensiveMore tanks requiredUseful only at early stagesBackground effects may obscuresome phenotypesRequires only 1 generationAll genomic regions accessibleMendelian ratiosBiased against telomeric regionsNon-Mendelian ratiosEP procedure reduces throughputBackground of EP effectsRequires only 1 generationDiploids screened: all stages accessible
62/3 of all mutants comprise 3 general phenotypic classes Widespread cell deathWhat types of genesmight be mutated?Heart edema/poor circulationGeneral “housekeeping” genes. What are they?Widespread cell deathstarting in CNSWild typeGeneral retardationin developmentWild type
8Zebrafish Spermatogenesis Spermatogonial stem cellENU induces mutations atall stages of spermatogenesis.Clones of mutations can occur,so keep track of F1s from each Founder male.Leal et al., 2009; Schulz et al., 2009