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
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!
Relative Attributes of Genetic Systems
Yeast
Forward
++
Drosophila
++
C. elegans
++
zebrafish
++
+/(difficult,
expensive)
(phenotype-based)
++
Reverse
(gene-based)
mouse
homologous
recombination
* Not really genetics
+
P-elements
RNAi*
homologous recomb.
+
RNAi*
Mutation Detection
+
Morpholinos*
Mutation Detection
ZFN, TALENs
++
homologous
recombination
Pros
Standard 3 generation
screen
Haploids
Gynogenetic diploids
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
Cons
Requires two generations
Labor-intensive
More tanks required
Useful only at early stages
Background effects may obscure
some phenotypes
Biased against telomeric regions
Non-Mendelian ratios
EP procedure reduces throughput
Background of EP effects
Zebrafish Zygotic Mutant Screen--Natural Crosses
G0
F1
*/+
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
Heart edema/poor circulation
Widespread cell death
starting in CNS
Wild type
General retardation
in development
Wild type
What types of genes
might be mutated?
General “housekeeping”
genes. What are they?
Zebrafish Zygotic Mutant Screen--Natural Crosses
G0
F1
*/+
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
F2
Gynogenetic diploid
Block 2nd polar
body formation
with ‘Early
Pressure’ (EP)
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/
• 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
G0
F1
*/+
F2
+/+
50%
*/+
50%
+/+
F3
Screen F3 embryos
morphologically at 1 dpf,
2 dpf, 5 dpf.
Zebrafish TILLING pipeline
(Targeted Induced Local Lesions in Genomes)
Mutation
detection (Cel1 or
Illumina)
library fertility over time
60%
percent fertility
50%
40%
30%
20%
10%
0%
2001
(n=11)
2002
(n=5)
2003
(n=8)
2004
(n=4)
2005
(n=13)
2006
(n=20)
2007
(n=24)
2008
(n=22)
2009
(n=19)
2010 (n8)
year
N=135