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Abstract
Grant Number: 1R43CA103649-01 PI Name: ADZUMA, KENJI PI Email: kenji@genetargeting.com PI Title: Project Title: New tool for insertional mutagenesis in the mouse Abstract: 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.
Thesaurus Terms:
biotechnology, laboratory mouse, mutant, technology /technique development, transgenic animal, transposon /insertion element
cell nucleus, egg /ovum
Institution: INGENIOUS TARGETING LAB, INC. 25 E LOOP RD, STE 106 STONY BROOK, NY 11790 Fiscal Year: 2003 Department: Project Start: 20-AUG-2003 Project End: 31-JUL-2004 ICD: NATIONAL CANCER INSTITUTE IRG: ZRG1