Efficient target-selected mutagenesis in zebrafish
Efficient target-selected mutagenesis in zebrafish
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DOI：
10.1101/gr.1725103
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发表时间：
2003-12-01
期刊：
影响因子：
7
通讯作者：
Cuppen, E
中科院分区：
文献类型：
--
作者：
Wienholds, E;van Eeden, F;Cuppen, E
为基因分配功能的最有效方法之一是删除或敲除该基因。最近，我们描述了第一个目标选择敲除斑马鱼。在这里，我们报告的进一步改进，这一程序，从而在一个高效和简单的方法来做靶向选择性诱变斑马鱼。生成4608只ENU诱变的F-1动物的文库，并作为活原种保存。通过使用CEL-I介导的异源双链体切割（TILLING）和随后的重测序来筛选这些动物的DNA中的16个基因中的突变。总共鉴定了255个突变，其中14个导致提前终止密码子，7个导致剪接供体/受体位点突变，119个导致氨基酸改变。通过这种方法，我们可能在几个月内敲除13个不同的基因。此外，我们表明，TILLING可用于检测在脊椎动物基因组中存在许多自然发生的多态性的ENU诱导的突变的全谱。
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 F-1 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.