| Co-Investigator(Kenkyū-buntansha) |
KATO Ichiro Toyama Medical & Pharmaceutical University, School of Medicine, Associate Professor, 医学部, 助教授 (50250741)
TAKASAWA Shin Tohoku University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (50187944)
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| Research Abstract |
Regenerating gene (Reg) encodes a secretory protein with a growth stimulating effect on pancreatic β cells. Reg and Reg-related genes have been revealed to constitute a multigene family which consists of three subtypes. We have isolated three types of mouse Reg family gene (J. Biol. Chem. 268, 15974, 1993, Gene 185, 159. 1997).
In the present study, by Southern blot analysis of a mouse BAC clone containing the five Reg family genes in combination with PCR cloning, the Reg family genes were mapped to a contiguous 75 kbp region on the mouse chromosome 6C/D to the following order: 5'-Reg IIIβ-Reg IlIα-Reg II-Reg I-Reg IIIγ-3'. In the process of ordering the genes, we sequenced the 6.8 kbp interspace fragment Reg IIIβ and Reg IIIα, and encountered a novel type III Reg gene, Reg IIIδ. This gene is divided into six exons spanning about 3 kbp, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg proteins.
To generate mutant mice deficent in all the Reg family
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gene, we constructed two targeting vectors. In one vector the loxP sequence was introduced into the 5'-upstream region of Reg IIIβ gene. In the other the loxP sequence was introduced into the 5'-upstream region of Reg IIIγ gene. Now the targeting vector was electroporated in ES cells.
Recently, a new member of human REG family gene, REG IV, was isolated. REG IV is located on chromosome 1 , while the other members are located on chromosome 2, suggesting a possibility that out of the cluster on chromosome 6C/D a new member of mouse REG family gene exist. On the other hand, we isolated a Reg receptor cDNA and the cells into which the cDNA has been introduced bound Reg I with high affinity (J. Biol. Chem. 275, 10723, 2000). The expression of Reg receptor mRNA was detected in various tissues including pancretaic islets and this receptor also bound to the Reg proteins other than Reg I. These results and the fact that there is no report about Reg receptor except for ours suggest the possibility that the receptor we isolated is the only recepor for Reg proteins. Therefore, we generate Reg receptor deficient mice. The targeting vector for Reg receptor disruption was electroporated in ES cells. Now the correctly targeted ES cells are aggregated with tetraploid embryos to produce germ-line chimeras. Less
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