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Round table discussion: Genetic Screens and Mutagenesis Methods 1)Mutagenesis Chemical Radiation Retroviruses and transposon insertional vectors Zinc finger.

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Presentation on theme: "Round table discussion: Genetic Screens and Mutagenesis Methods 1)Mutagenesis Chemical Radiation Retroviruses and transposon insertional vectors Zinc finger."— Presentation transcript:

1 Round table discussion: Genetic Screens and Mutagenesis Methods 1)Mutagenesis Chemical Radiation Retroviruses and transposon insertional vectors 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, zinc finger nucleases, TALENs

2  -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

3 Relative Attributes of Genetic Systems Yeast Drosophila C. eleganszebrafishmouse Forward Reverse ++ +/- (difficult, expensive ) +++++ homologous recombination homologous recombination P-elements RNAi* homologous recomb. RNAi* Mutation Detection Morpholinos* Mutation Detection ZFN, TALENs (gene-based) (phenotype-based) * Not really genetics

4 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

5 Zebrafish Zygotic Mutant Screen--Natural Crosses G0 F1 F2 F3 Screen F3 embryos morphologically at 1 dpf, 2 dpf, 5 dpf. */+*/+ +/+ 50% */ + 50% +/+

6 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 What types of genes might be mutated? General “housekeeping” genes. What are they?

7 Zebrafish Zygotic Mutant Screen--Natural Crosses G0 F1 F2 F3 Screen F3 embryos morphologically at 1 dpf, 2 dpf, 5 dpf. */+*/+ +/+ 50% */ + 50% +/+

8 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

9 F1 F2

10 Early pressure (diploid) Block 2nd polar body formation with ‘Early Pressure’ (EP) Gynogenetic diploid F1 F2

11 Reverse Genetic Approaches in Zebrafish 1)ANTISENSE (MORPHOLINOS) 2)MUTATION DETECTION TILLING: i) whole genome (Illumina): “Zebrafish Mutation Project” (Stemple): http://www.sanger.ac.uk/Projects/D_rerio/zmp/ ii) Gene-by-gene: “Zebrafish TILLING Consortium” (Moens, Solnica-Krezel): https://webapps.fhcrc.org/science/tilling/ https://webapps.fhcrc.org/science/tilling/ Retroviral mutagenesis (Burgess, Lin) Wang et al. (2007) Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. PNAS 104: 12428 (e.g. sfrp1a)sfrp1a 3) TARGETED MUTAGENESIS Zinc-finger nucleases Sander et al. (2011) selection-free Zn-finger nuclease engineering by context-dependent assembly (CoDA) TALENs

12 Zebrafish Zygotic Mutant Screen--Natural Crosses G0 F1 F2 F3 Screen F3 embryos morphologically at 1 dpf, 2 dpf, 5 dpf. */+*/+ +/+ 50% */ + 50% +/+

13 Zebrafish TILLING pipeline (Targeted Induced Local Lesions in Genomes) Mutation detection (Cel1 or Illumina)

14 N=135

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