New tool for insertional mutagenesis in the mouse

描述（由申请人提供）：本提案的长期目标是创建一个随机突变小鼠库，每个突变小鼠都有一个插入突变。这样的文库对于功能分析和筛选与人类疾病相关的小鼠基因将非常有用。我们实现这一目标的计划是：(1)在试管中，在转座子DNA分子和催化转座子的酶之间形成复合物，然后(2)将这种具有催化能力的DNA-酶复合物直接注射到受精卵的前核中。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。这种方法无需通过胚胎干细胞（ES）就能产生突变小鼠。因此，与首先使胚胎干细胞发生突变，然后从胚胎干细胞中产生突变小鼠的传统插入突变策略相比，这种方法更快，劳动强度更低。

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.