ROLES OF THE MFH-1 AND WT1 GENES IN KIDNEY DEVELOPMENT

MFH-1 和 WT1 基因在肾脏发育中的作用

基本信息

批准号：
09044252
负责人：
MIURA Naoyuki
金额：
$ 1.86万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044252/
关键词：
FORKHEAD GENES MFH-1 GENE KNOCKOUT MICE WILMS TUMOR KIDNEY WT1 GENE TARGET GENES HIERARCHY ウイルムス腫瘍尿細管糸球体分化 PAM染色

项目摘要

We generated the knockout mice of the MIFH-1 gene. The MIFH-1 null mice displayed phenotypes of cardiovascular system and skeleton. The formers were interruption of the aortic arch and ventral septal defect, and the latters were defects arid malformation of the vertebrates and craniofacial bones. These results indicate that the MFH-1 gene is essential for aortic arch formation and skeletogenesis.The MFH-1 gene is also expressed in the kidney. In the MFH-1 knockout mice, the kidneys were a little small and the pelvis was malformed. However, the structures of glomerulus and renal tubules had no abnormalities. We interpret that other forkhead family genes, for example BF-1 and MF-1, might compensate the function of the MFH-1 gene.Next we investigated the target genes of MFH-1 gene. We screened the cell lines with monoclonal anti-mouse MFH-1 antibody in order to study whether the MFH-1 gene is expressed in the cells. We found that the MFH-1 protein was expressed in the Wilms tumor cell line, osteosarcoma cells, chondrosarcoma cells, aortic endothelial cells, aortic smooth muscle cells and fibroblast cells, NIH/3T3 and Balb/3T3. In order to investigate the molecular mechanism governing the transcriptional control of the MFH-1 gene in the kidney, we transfected the 3.Okbp of promoter and 5'-flanking region of the MFH-1 gene which was connected to the reporter gene luciferase into the Wilms tumor cell line. The construct which contains 3.0kbp flanking region showed a strong transcriptional activity in the Wilms tumor cell line. This result indicates that the 3.0 kbp region has elements regulating the expression of the MFH-1 gene positively in the nephroblastoma cells. Currently, the WT1-binding sites within the 3.0kbp region have been tried to identify by gel shift assay and cotransfection with the WT1-expression vector.

我们生成了MIFH-1基因的敲除小鼠。 MIFH-1 NULL小鼠显示了心血管系统和骨骼的表型。造型器是主动脉弓和腹膜间隔缺陷的中断，后者是脊椎动物和颅骨骨骼干旱畸形的缺陷。这些结果表明，MFH-1基因对于主动脉弓的形成和骨骼生成至关重要。MFH-1基因在肾脏中也表达。在MFH-1敲除小鼠中，肾脏有点小，骨盆被畸形。但是，肾小球和肾小管的结构没有异常。我们解释说，其他叉状家族基因（例如BF-1和MF-1）可能会补偿MFH-1基因的功能。我们研究了MFH-1基因的靶基因。我们用单克隆抗小鼠MFH-1抗体筛选了细胞系，以研究MFH-1基因是否在细胞中表达。我们发现MFH-1蛋白在Wilms肿瘤细胞系，骨肉瘤细胞，软骨肉瘤细胞，主动脉内皮细胞，主动脉平滑肌细胞和成纤维细胞细胞，NIH/3T3和BALB/3T3中表达。为了研究肾脏中MFH-1基因转录控制的分子机制，我们转染了MFH-1基因的启动子的3.okbp和5'-fanking区域，该基因与报告基因荧光素酶连接到WILMS TUMOR细胞系。包含3.0kbp侧翼区域的构建体在Wilms肿瘤细胞系中表现出很强的转录活性。该结果表明3.0 KBP区域具有调节MFH-1基因在肾细胞瘤细胞中的表达的元素。当前，已尝试通过WT1-Expression载体通过凝胶移位分析和共转染来识别3.0kbp区域内的WT1结合位点。