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)