Target-Selected Inactivation of the Zebrafish rag1 Gene

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 muta...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2002-07, Vol.297 (5578), p.99-102
Main Authors: Wienholds, Erno, Schulte-Merker, Stefan, Walderich, Brigitte, Ronald H. A. Plasterk
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Biological and medical sciences
Coding
Codon, Terminator
Danio rerio
Developmental Stages
Ethylnitrosourea
Female
Fish
Freshwater
Fundamental and applied biological sciences. Psychology
gene inactivation
Gene Library
Gene Rearrangement
Genes
Genes, Immunoglobulin
Genes, RAG-1
Genetic aspects
Genetic mutation
Genetic screening
Genetics
Genomes
Genomics
Haplotypes
Heterozygote
Homeodomain Proteins - chemistry
Homeodomain Proteins - genetics
Immunoglobulin Heavy Chains - genetics
Introns
Libraries
Male
Marine fishes
Molecular and cellular biology
Molecular genetics
Mutagenesis
Mutagenesis. Repair
Mutation
Mutation, Missense
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
rag1 gene
Recombination, Genetic
Reverse genetics
Sarcoma
Tumors
Zebra fish
Zebrafish
Zebrafish - genetics
Zebrafish - immunology
Zebrafish - physiology
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Summary: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 F1fish, 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 F1fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.1071762