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Science 5 July 2002:
Vol. 297. no. 5578, pp. 99 - 102
DOI: 10.1126/science.1071762
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Reports
Target-Selected Inactivation of the Zebrafish rag1 Gene
Erno Wienholds,1
Stefan Schulte-Merker,2
Brigitte Walderich,2
Ronald H. A. Plasterk1*
The zebrafish has become a favorite organism for genetic
analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to
obtain stable mutants of a gene based on knowledge of the gene sequence
only. Parental fish were mutagenized with
N-ethyl-N-nitrosourea; in 2679 F1
fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop
codon. This truncation mutant was found to be homozygous viable and
defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and
are fertile. As sperm samples from all 2679 F1 fish were
collected and cryopreserved, we have in principle generated a mutant
library from which mutants of most zebrafish genes can be isolated.
1 Hubrecht Laboratory, Center for Biomedical
Genetics, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands.
2 ARTEMIS Pharmaceuticals GmbH/ Exelixis
Deutschland, Spemannstrasse 35, 72076 Tübingen, Germany.
*
To whom correspondence should be addressed. E-mail:
plasterk{at}niob.knaw.nl.
Read the Full Text
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