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Targeted disruption of all the Reg family genes: A new animal model for regeneration deficient disorders

有针对性地破坏所有 Reg 家族基因：再生缺陷性疾病的新动物模型

基本信息

批准号：
11557009
负责人：
NATA Koji
金额：
$ 8.58万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557009/
关键词：
Reg Pancreatic isletβ-cells Regeneration Cre-lox P system Diabetes mellitus Regenerating gene gene family BAC clone Cre-lox Pシステム Cre-lox P システム

项目摘要

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 … More 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

再生基因（Reg）编码一种对胰腺β细胞具有生长刺激作用的分泌蛋白。Reg和Reg相关基因组成了一个多基因家族，由三种亚型组成。我们已经分离了三种类型的小鼠Reg家族基因（J. Biol. Chem. 268，15974，1993，Gene 185，159.在本研究中，通过Southern印迹分析含有5个Reg家族基因的小鼠BAC克隆并结合PCR克隆，将Reg家族基因定位到小鼠染色体6C/D上的连续75 kbp区域，顺序如下：5 '-Reg IIIβ-Reg III α-Reg II-Reg I-Reg IIIγ-3'。在基因排序过程中，我们对6.8kbp的间隔片段Reg IIIβ和Reg IIIα进行了测序，发现了一个新的III型Reg基因Reg IIIδ。该基因由6个外显子组成，长度约为3 kbp，编码一个175个氨基酸的蛋白质，与其它5个小鼠Reg蛋白的同源性为40-52%。 ...更多信息基因，我们构建了两个靶向载体。在一个载体中，将loxP序列引入Reg IIIβ基因的5 '上游区域。另一种是将loxP序列引入Reg IIIγ基因的5 '上游区域。目前已成功地将其电穿孔到ES细胞中，并分离到了人REG家族的一个新成员--REG IV。REG IV位于1号染色体上，而其他成员位于2号染色体上，这表明在6C/D染色体上的簇中可能存在小鼠REG家族基因的新成员。另一方面，我们分离了Reg受体cDNA，并且已导入cDNA的细胞以高亲和力结合Reg I（J. Biol. Chem. 275，10723，2000）。在包括胰岛在内的各种组织中检测到Reg受体mRNA的表达，并且该受体也结合除了Reg I之外的Reg蛋白。这些结果以及除了我们的研究外，目前还没有关于Reg受体的报道的事实表明，我们分离的受体可能是Reg蛋白的唯一受体。因此，我们产生了Reg受体缺陷型小鼠。在ES细胞中电穿孔用于Reg受体破坏的靶向载体。现在，正确靶向的ES细胞与四倍体胚胎聚集以产生生殖系嵌合体。少