PUBLICATION
Efficient target-selected mutagenesis in zebrafish
- Authors
- Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R.H., and Cuppen, E.
- ID
- ZDB-PUB-031119-10
- Date
- 2003
- Source
- Genome research 13: 2700-2707 (Journal)
- Registered Authors
- Cuppen, Edwin, Plasterk, Ronald H.A., van Eeden, Freek, Wienholds, Erno
- Keywords
- none
- MeSH Terms
-
- Animals
- Crosses, Genetic
- DNA Primers/chemical synthesis
- Drug Administration Schedule
- Ethyl Methanesulfonate/pharmacology
- Female
- Gene Library
- Male
- Mutagenesis, Site-Directed*
- Nucleic Acid Amplification Techniques
- Nucleic Acid Heteroduplexes/metabolism
- Plant Proteins/metabolism
- Point Mutation
- Zebrafish/genetics*
- PubMed
- 14613981 Full text @ Genome Res.
Citation
Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R.H., and Cuppen, E. (2003) Efficient target-selected mutagenesis in zebrafish. Genome research. 13:2700-2707.
Abstract
One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently, we described the first target-selected knockout in zebrafish. Here, we report on the further improvements of this procedure, resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total, 255 mutations were identified, of which 14 resulted in a premature stop codon, 7 in a splice donor/acceptor site mutation, and 119 in an amino acid change. By this method, we potentially knocked out 13 different genes in a few months time. Furthermore, we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping