Round table discussion: Genetic Screens and Mutagenesis Methods • 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 ADD spermatogenesis stage to explain mutating sperm v various spermatogonial stages. 2011

Mutagens g-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 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!

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

Cons Pros Standard 3 generation screen Haploids Gynogenetic diploids All stages and genomic regions accessible Mendelian ratios Natural breeding is the only “technique” Standard 3 generation screen Haploids Gynogenetic diploids Requires two generations Labor-intensive More tanks required Useful only at early stages Background effects may obscure some phenotypes Requires only 1 generation All genomic regions accessible Mendelian ratios Biased against telomeric regions Non-Mendelian ratios EP procedure reduces throughput Background of EP effects Requires only 1 generation Diploids screened: all stages accessible

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

2/3 of all mutants comprise 3 general phenotypic classes Widespread cell death What types of genes might be mutated? Heart edema/poor circulation General “housekeeping” genes. What are they? Widespread cell death starting in CNS Wild type General retardation in development Wild type

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

Zebrafish Spermatogenesis Spermatogonial stem cell ENU induces mutations at all 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

F1 F2

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

Reverse Genetic Approaches in Zebrafish ANTISENSE (MORPHOLINOS) 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/ • Retroviral mutagenesis (Burgess, Lin) Wang et al. (2007) Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. PNAS 104: 12428 (e.g. sfrp1a) 3) TARGETED MUTAGENESIS • Zinc-finger nucleases Sander et al. (2011) selection-free Zn-finger nuclease engineering by context-dependent assembly (CoDA) • TALENs

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

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

N=135