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

再生基因（Reg）编码一种分泌蛋白，对胰腺β细胞具有刺激生长的作用。regg和regg相关基因组成了一个多基因家族，由三个亚型组成。我们分离了三种类型的小鼠Reg家族基因（J. Biol.）。化学268,15974,1993，基因185,159。1997)。本研究通过对含有5个Reg家族基因的小鼠BAC克隆进行Southern blot分析，结合PCR克隆，将Reg家族基因定位到小鼠6C/D染色体上连续75 kbp的区域，顺序为：5′-Reg IIIβ-Reg il α-Reg II-Reg I-Reg IIIγ-3′。在基因排序过程中，我们测序了6.8 kbp的间隙片段Reg IIIβ和Reg IIIα，并遇到了一个新的III型Reg基因Reg IIIδ。该基因被分成6个长约3 kbp的外显子，编码一个175个氨基酸的蛋白，与其他5个小鼠Reg蛋白具有40-52%的同源性。为了获得Reg家族…More全部基因缺失的突变小鼠，我们构建了两个靶向载体。在一个载体上，将loxP序列导入Reg IIIβ基因的5′上游区域。另一种方法是将loxP序列引入到Reg iii - γ基因的5'上游区域。现在靶向载体在胚胎干细胞中电穿孔。最近，人类REG家族基因的一个新成员REG IV被分离出来。REG IV位于1号染色体上，而其他成员位于2号染色体上，提示在6C/D染色体簇外可能存在小鼠REG家族基因的新成员。另一方面，我们分离了一个Reg受体cDNA，并将该cDNA引入细胞中以高亲和力结合Reg I (J. Biol。化学，275,10723,2000)。在包括胰岛在内的多种组织中检测到Reg受体mRNA的表达，并且该受体还与Reg i以外的Reg蛋白结合。这些结果以及除了我们的报道外没有关于Reg受体的报道，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regg受体缺陷小鼠。在胚胎干细胞中电穿孔介导Reg受体破坏的靶向载体。现在，正确靶向的胚胎干细胞与四倍体胚胎聚集在一起，产生种系嵌合体。少

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