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 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.
Wienholds
Erno
E
Hubrecht Laboratory, Center for Biomedical Genetics, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands.
Schulte-Merker
Stefan
S
Walderich
Brigitte
B
Plasterk
Ronald H A
RH
D019943:Amino Acid Substitution; D000818:Animals; D018388:Codon, Terminator; D005038:Ethylnitrosourea; D005260:Female; D015723:Gene Library; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D016654:Genes, RAG-1; D006239:Haplotypes; D006579:Heterozygote; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007438:Introns; D008297:Male; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D015027:Zebrafish / Q000235:genetics* / Q000276:immunology / Q000502:physiology
D018388:Codon, Terminator; D018398:Homeodomain Proteins; D007143:Immunoglobulin Heavy Chains; C064658:RAG-1 protein; D005038:Ethylnitrosourea