New tool for insertional mutagenesis in the mouse

小鼠插入突变的新工具

• 批准号: 6583036
• 负责人: KENJI ADZUMA
• 金额: $ 9.95万
• 依托单位: INGENIOUS TARGETING LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-20 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6583036
• 关键词: biotechnology; cell nucleus; egg /ovum; genetically modified animals; laboratory mouse; mutant; technology /technique development; transposon /insertion element

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to create a random library of mutant mice, each with an insertional mutation. Such library would be enormously useful for functional analyses as well as screening of the mouse genes relevant to human diseases. Our plan to achieve this goal is: (1) in a test tube, form a complex between a transposon DNA molecule and the enzymes that catalyze the transposition, and then (2) inject this catalytically competent DNA-enzyme complex directly into a pro-nucleus of fertilized mouse egg. If the insertion is successful, mutant pups will be born only three weeks later. As the transposon, we will use phage Mu system. This method creates mutant mice without going through Embryonic Stem (ES) cells. It is therefore much quicker and less labor-intensive than the conventional strategy of insertional mutagenesis in which one first mutagenizes ES cells and then creates mutant mice from the ES cells. During this phase I period, our primary objective is to actually try this method, in order to assess the feasibility of our idea as well as to address various technical issues that may arise. Thus the specific aims are: (a), We will inject the Mu transposon complex into a pro-nucleus of mouse egg and analyze the insertion efficiency by Southern blotting and/or PCR analyses of the DNA from the offspring. (b), To distinguish Mu-dependent insertion from random integration of naked DNA, we will clone and sequence the insertion junctions; Mu transposition should create a characteristic 5-nucleotide duplicate at the junctions.

描述（由申请人提供）：本提案的长期目标是创建突变小鼠的随机文库，每个突变小鼠具有插入突变。该文库对于功能分析和筛选与人类疾病相关的小鼠基因具有重要意义。我们实现这一目标的计划是：（1）在试管中，在转座子DNA分子和催化转座的酶之间形成复合物，然后（2）将这种具有催化活性的DNA-酶复合物直接注入受精小鼠卵的原核中。如果插入成功，突变的幼崽将在三周后出生。作为转座子，我们将使用噬菌体Mu系统。这种方法不需要通过胚胎干细胞（ES细胞）就能产生突变小鼠。因此，它比传统的插入诱变策略更快，劳动强度更低，在传统的插入诱变策略中，首先诱变ES细胞，然后从ES细胞产生突变小鼠。 在第一阶段期间，我们的主要目标是实际尝试这种方法，以评估我们的想法的可行性，并解决可能出现的各种技术问题。因此，具体目标是： (a)我们将Mu转座子复合物注射到小鼠卵细胞的原核中，并通过Southern印迹和/或PCR分析后代DNA来分析插入效率。 (b)为了区分Mu依赖性插入与裸DNA的随机整合，我们将克隆和测序插入接头; Mu转座应在接头处产生特征性的5个核苷酸重复。