Presentation on theme: "Round table discussion: Genetic Screens and Mutagenesis Methods"— Presentation transcript:
1 Round 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
2 Mutagensg-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
4 Cons 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
6 2/3 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
8 Zebrafish 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