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Published online before print November 12, 2003, 10.1101/gr.1725103
Genome Res. 13:2700-2707, 2003
©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
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Methods

Efficient Target-Selected Mutagenesis in Zebrafish

Erno Wienholds, Freek van Eeden, Marit Kosters, Josine Mudde, Ronald H.A. Plasterk and Edwin Cuppen1

Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

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.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1725103. Article published online before print in November 2003.

1 Corresponding author.
E-MAIL ecuppen{at}niob.knaw.nl; FAX 31-30-2516554.

[Supplemental material is available online at www.genome.org.]


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