Efficient target-selected mutagenesis in zebrafish

Genome Res. 2003 Dec;13(12):2700-7. doi: 10.1101/gr.1725103. Epub 2003 Nov 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

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*

Substances

  • DNA Primers
  • Nucleic Acid Heteroduplexes
  • Plant Proteins
  • Ethyl Methanesulfonate