Efficient target-selected mutagenesis in zebrafish

E. Wienholds, F. van Eeden, M. Kosters, J.B. Mudde, R. Plasterk, E. Cuppen

Research output: Contribution to journal/periodicalArticleScientificpeer-review

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.
Original languageEnglish
Pages (from-to)2700-2707
JournalGenome Research
Volume13
Issue number12
DOIs
Publication statusPublished - 2003

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