International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)

Table of Contents * Complex Genetics * Developmental Genetics * Gene Annotation * Gene Discovery * Genome Sequencing * Functional Genomics * Mutagenesis * Presentations * Verne Chapman Memorial Lecture

Mr Masatake Araki
Gene Technology Centre
Kumamoto University
2-2-1, Honjo
Kumamoto 860-0811 Japan

Co-Authors: 1)Yoshinobu K, 2)Taniwaki T, 2)Sekimoto A, 2)Haruna K, 2)Oike Y, 2)Imaizumi T, 2)Yoshimuta J, 2)Akizuki M, 2)Miura K, 2)Li Z, 2)Senba K, 3)Suzuki M, 3)Nakagata N, 4)Saito F, 5)Niizato H, 5)Nakashima T, 5)Muta M, 6)Utou A, 6)Kimura Y, 6)Sakumura Y, 6)Yanagihara S, 6)Nakatsukasa E, 6) Tanaka M, 6)Kagami Y, 2)Yamamura K, 2)Araki K
Institutions: 1)GTC, Kumamoto University, 2)IMEG, Kumamoto Univerasity, 3)CARD, Kumamoto University, 4)Chemical Evaluation and Technology Corporation, 5)Japan Science and Technology Corporation, 6)Transgenic Inc

Exchangeable gene trapping vector with or without IRES, We have performed the exchangeable gene trap using Cre-mutant lox system.   this work, we present two kinds of trapping vector, pU-Hachi and pU-17, and their integration points in trapped genes.

In both of the vectors, the splice acceptor (SA) of the mouse En-2 gene and the beta-geo gene are used.  Although the positions and kinds of the lox sites used in the vectors are different, the most important difference affecting the efficiency of trapping event is the structure around the start codon of the beta-geo. In pU-Hachi, the internal ribosomal entry site (IRES) is used.  This allows efficient trapping independent from the integration point in trapped genes.  On the other hand, in pU-17, just a simple ATG is used and an in-frame stop codon presents in 24-bp upstream of the ATG.  Therefore, it is theoretically expected that the insertion points should be restricted in upstream of the start codon of trapped gene but be able to induce null mutation effectively.

The vectors were introduced into TT2 ES cells through electroporation, and the clones having a single copy of the vector integrated were selected and used for production of chimeric mice.  Until now, we have established 26 and 50 mutant mouse lines using pU-hachi and pU-17, respectively.

Analyses of the 5'RACE revealed that the integration point of pU-17 showed a tendency to be concentrated within 200 bp of the start codon of trapped gene, whereas the integration point of pU-Hachi was random.