International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 213 - HANGEABLE GENE TRAP USING A "DOUBLE LOX" TARGETING STRATEGY MEDIATED BY CRE

Dr Kimi Araki
IMEG, Kumamoto University
4-24-1 Kuhonji
Kumamoto 860-0976 Japan

Co-Authors: 2)Araki M, 1)Haruna K, 3)Muta M, 4)Kimura Y, 1)Yamamura K
Institutions: 1)IMEG Kumamoto University, 2)GTC Kumamoto University, 3)Japan Science & Technology Corporation, 4)Transgenic Inc

Exchangeable gene trap using a "double lox" targeting strategy mediated by cre.  The gene trap technique is a powerful approach to characterize and mutate genes.  However, one shortcoming of gene trapping is its relative inability to induce subtle or gain-of-function mutations.  To overcome this problem, we have developed Cre-mutated lox system in which a pair of mutant lox, lox71 and lox66, was used to promote integrative reaction by Cre recombinase, and applied this system for gene trap.

To improve the applicability of trap vector more, we have decided to use "double lox" system, in which a heterospesific lox site that does not recombine with the wild type loxP but itself, are utilized.  Since several heterospesific lox sequences have been reported, we chose two lox sequences, lox511 and lox2272, compared their recombination efficiencies in ES cells, and found that lox2272 is more efficient than lox511.

Furthermore, taking the advantage of this system in which the recombined structure is stable even if the Cre recombinase is exist, we successfully introduced cre gene into lox sites by Cre-mediated recombination.

Now, we have constructed a new trap vector pU-17 using lox71 and lox2272, and established more than 1000 trap clones and 50 mutant mouse lines. By using this vector, we can carry out random mutagenesis in the first step, and then replace the beta-geo gene into any other gene of interest (including cre gene) to have expressed under the control of the promoter of the trapped gene through Cre-mediated integration.


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