ZFIN ID: ZDB-PUB-031119-10
Efficient target-selected mutagenesis in zebrafish
Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R.H., and Cuppen, E.
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.
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.