Wienholds Erno, Schulte-Merker Stefan, Walderich Brigitte, Plasterk Ronald H A
Hubrecht Laboratory, Center for Biomedical Genetics, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands.
Science. 2002 Jul 5;297(5578):99-102. doi: 10.1126/science.1071762.
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.
斑马鱼已成为脊椎动物发育基因分析中备受青睐的生物体，但一直缺乏通过反向遗传学方法产生突变体的手段。我们报道了一种仅基于基因序列知识来获得基因稳定突变体的方法。用N-乙基-N-亚硝基脲对亲代鱼进行诱变；在2679条F1代鱼中，通过重测序分析rag1基因的杂合突变。我们总共发现了15个突变：9个导致氨基酸替换，1个导致提前终止密码子。发现这种截短突变体纯合子可存活且在V(D)J连接方面存在缺陷。尽管可能存在免疫缺陷，但这些纯合rag1突变体鱼能够成年并可育。由于收集并冷冻保存了所有2679条F1代鱼的精子样本，我们原则上已构建了一个突变体文库，从中可分离出大多数斑马鱼基因的突变体。
