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 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

2. 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!

3. +/-(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

4. 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

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

6. 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

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

8. 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

9. F1 F2

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

11. Reverse Genetic Approaches in Zebrafish
ANTISENSE (MORPHOLINOS)
MUTATION DETECTION
• TILLING:
i) whole genome (Illumina): “Zebrafish Mutation Project” (Stemple):
ii) Gene-by-gene: “Zebrafish TILLING Consortium” (Moens, Solnica-Krezel):
• Retroviral mutagenesis (Burgess, Lin)
Wang et al. (2007) Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. PNAS 104: (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

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

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

14. N=135