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Roles of the MFH-1 gene and WT 1 gene in kidney development

MFH-1 基因和 WT 1 基因在肾脏发育中的作用

基本信息

批准号：
08044238
负责人：
MIURA Naoyuki
金额：
$ 1.02万
依托单位：
Akita university School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044238/
关键词：
MFH-1 gene Knockout mice Kidney development monoclonal antibody MFH-1 protein

项目摘要

We isolated the MFH-1 gene as one of the forkhead gene family in 1993. The family are growing and more than 20 genes are known in mice. The MFH-1 gene is expressed at the somitic mesoderm and later expressed strongly in the sclerotome. It is also expressed in the developing metanephros and later on in the glomerulus after birth.We made knockout mice of the MFH-1 gene by homologous recombination in the ES cells. First, we cloned the mouse genomic MFH-1 gene and determined its genomic structure. We found that it has no introns. We made a targeting vector by replacing the exon with neo-resistance cassette and introduced it into ES cells by electroporation. Out of 480 neomycin- and gancyclovir-resistant clones, 6 were identified to be homologous recombinants. We made chimera mice by injecting them into a blastocyst and obtained heterozygotic mice by mating chimera mice and wild B6 mice. The heterozygotes were asymptomatic. However, homozygotes were dead within ten minutes after birth.Homozygotes had abnormalities in several tissues. Here we focused the phenotype of kidneys. The size of kidneys in homozygotes was half that of the wild-type. Histologically the glomerulus, renal tubular cells and blood vesssels were present normally. But dilated renal calces were seen in homozygotes.Next, we cloned the human genomic MFH-1 gene and determined its nucleotide sequence. We found that human MFH-1 protein is 501 amino acids long and the amino acid sequence of the forkheah domain is identical in human and mouse. Identity of the mouse and human MFH-1 protein is 94%.We also made monoclonal antibodies against the human MFH-1 protein. Using thses antibodies, we found that the MFH-1 protein is expressed in chondrosarcoma, osteosarcoma and Wilms tumor cell lines.

我们于1993年分离到MFH-1基因作为叉头基因家族的一员。这个家族还在不断壮大，目前已知老鼠体内有20多个基因。MFH-1基因在体裂中胚层表达，随后在硬核组强烈表达。它也在发育中的后肾和出生后的肾小球中表达。我们在胚胎干细胞中通过同源重组制备MFH-1基因敲除小鼠。首先，我们克隆了小鼠基因组MFH-1基因并确定了其基因组结构。我们发现它没有内含子。我们用新抗性盒代替外显子制成靶向载体，并通过电穿孔将其导入ES细胞。在480个新霉素和更昔洛韦耐药克隆中，6个被鉴定为同源重组。我们将嵌合体小鼠注射到囊胚中制备，并将嵌合体小鼠与野生B6小鼠交配得到杂合子小鼠。杂合子无症状。然而，纯合子在出生后10分钟内死亡。纯合子在一些组织中有异常。这里我们关注的是肾脏的表型。纯合子的肾脏大小是野生型的一半。组织学上肾小球、肾小管细胞和血管正常。但在纯合子中可见扩张的肾盏。接下来，我们克隆了人类基因组MFH-1基因并确定了其核苷酸序列。结果表明，人MFH-1蛋白全长501个氨基酸，其forkheah结构域的氨基酸序列与小鼠相同。小鼠和人MFH-1蛋白的同源性为94%。我们还制备了针对人MFH-1蛋白的单克隆抗体。利用这些抗体，我们发现MFH-1蛋白在软骨肉瘤、骨肉瘤和Wilms肿瘤细胞系中表达。