International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

H15 Random Mutagenesis in Mice Using the Exchangeable Gene Trap

Ken-ichi Yamamura1, Masatake Araki2, Takashi Imaizumi1, Tomohisa Sekimoto1, Kimi Araki1. 1Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kuhonji 4-24-1, Kumamoto 862-0976, Japan; 2Gene Technology Center, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan

With advances in human genome project, the structural analyses of human genome will be completed by the beginning of the 21st century. However, nucleotide sequences do not give sufficient information about the biological functions of genome. Thus, we need another approaches to analyze the biological functions. One of the promising approaches is the gene trap mutagenesis. However, one shortcoming of gene trapping is its relative inability to induce subtle mutations, such as point mutation. The other shortcoming is that the gene trap is more laborious than mutagenesis by chemical mutagen to establish mutant strains. To overcome the first problem, we have developed Cre-mutated lox system (Araki, K., Araki, M. & Yamamura, K. Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acid Res. 25:868-872, 1997) in which a pair of mutant lox, lox 71 and lox66, was used to promote integrative reaction by Cre recombinase. We have applied this system to create the new gene trap vector, pU-Hachi. This is composed of splicing acceptor (SA)-Internal ribosomal entry site (IRES)- lox 71-beta geo-pA- loxP-pA-pUC. Using this vector, we can carry out random mutagenesis in the first step. Then, we can replace the beta-geo gene with the gene of interest through Cre-mediated integration. We have isolated 109 trap clones and those clones carrying the single copy of trap vector were selected. Using the clones, we succeeded to exchange the reporter gene, beta-geo, with other gene such as EGFP at high efficiency. To address the second problem, we decided to use TT2 ES cells because we can make chimeric embryos by aggregating TT2 ES cells with morulae obtained from ICR strain. Using this method we can produce 240 lines of chimeric mice per person per year.

 


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