Construction of model mouse of the Beckwith-wiedemann syndrome

Beckwith-wiedemann综合征小鼠模型的构建

• 批准号: 11670145
• 负责人: MUKAI Tsunehiro
• 金额: $ 2.3万
• 依托单位: Saga Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670145/
• 关键词: genomic imprinting; p57Kip2 mutant; transgenic mouse; Beckwith-Wiedemann syndrome; インプリンティング; p57KIP2変異体; ゲノムインプリンティング; P57KIP2; アデノウイルスベクター

The CDK inhibitor, p57^<KIP2> is one of the gene responsible for Beckwitn-Wiedemann syndrome (BWS) of which locus is located on chromosome 11p15.5. To confirm the phenotype of this mutant at the cellular level and the animal, 1) we transfected the mutant to the cell and observed the biochemical and morphorogical effect, 2) and also created the transgenic mouse as a model of BWS.Adenovirus vector carrying mutant p57^<KIP2> cDNA was transfected to the G401 and RD cells. These cells do not express the endogenous p57KIP2. The mutant is Ad/KIP2-1-8 (mutaion at the C-ter of p57^<KIP2>). Controls are Ad/KIP2-1 (wild p57^<KIP2>) and Ad/LacZ.RD cell transfected with wild p57^<KIP2> cDNA expressed the protein. This was confirmed by Western blotting and by the staining with p57KIP2 antibody. However, the specific repression of the cell growth was not observed by the wild p57^<KIP2>. The level of the repression was similar with that of the mutant p57^<KIP2>. Next, we tried to create the transgenic mouse carrying mutant p57^<KIP2> as animal model to mimic BWS.pRc/CVM plasmid vector was used to obtain the high expression of human cDNA recombinant. These include pDR2-1 (wild), pDR2-1-6 (mutation of the CDK inhibitory domain) and pDR2-1-8 (mutaion at the QT domain). The obtained result showed all mice born were non-transgenic, suggesting the overexpression of this gene might be toxic. Conditional transgenics such as Cre/Lox system will be necessary to overcome this situation in the future.

CDK抑制因子p57是<KIP2>Beckeli-Wiedemann综合征（Beckeli-Wiedemann syndrome，BWS）的致病基因之一，其基因座位于染色体11p15.5。为了在细胞水平和动物体内证实该突变体的表型，我们将该突变体转染到细胞中，观察其生化和形态学效应，并建立了转基因小鼠作为BWS模型<KIP2>。这些细胞不表达内源性p57 KIP 2。突变体为Ad/KIP 2 -1-8（突变于p57的C端<KIP2>）。对照组为Ad/KIP 2 -1（野生型p57 ~+<KIP2>）和Ad/LacZ。转染野生型p57 ~+ cDNA的RD细胞<KIP2>表达蛋白。这通过蛋白质印迹和用p57 KIP 2抗体染色来证实。然而，未观察到野生型p57对细胞生长的特异性抑制<KIP2>。抑制水平与突变型p57相似<KIP2>。其次，我们尝试建立携带突变型p57^的转基因小鼠<KIP2>作为动物模型来模拟BWS，并利用pRc/CVM质粒载体获得高表达的人cDNA重组体。这些包括pDR 2 -1（野生型）、pDR 2 -1-6（CDK抑制结构域突变）和pDR 2 -1-8（QT结构域突变）。结果表明，所有出生的小鼠均为非转基因小鼠，提示该基因的过表达可能具有毒性。有条件的转基因如Cre/Lox系统将是克服这一问题的必要手段。

项目成果

Zhu,X.: "Cllorf21, a novel gene within the Beckwith-Wiedemann syndrime region in human chromosome 11p15.5"Gene. 256. 311-317 (2000)

Zhu, X.：“Cllorf21，人类染色体 11p15.5 Beckwith-Wiedemann 综合征区域内的一个新基因”基因。

Bhuiyan ZA.: "Functional analysis of the p57KIP2 gene mutation in Beckwith-Wiedemann syndrome."Human Genet.. 104. 205-210 (1999)

Bhuiyan ZA.：“Beckwith-Wiedemann 综合征中 p57KIP2 基因突变的功能分析。”Human Genet.. 104. 205-210 (1999)

Higashimoto K.: "Identification of a novel single nucleotide porimorphism (SNP) in the human organic cation transporte-like 2-antisense (ORCTLTS) gene"J Human Genet. 45. 58-59 (2000)

Higashimoto K.：“人类有机阳离子转运样 2-反义 (ORCTLTS) 基因中新型单核苷酸多态性 (SNP) 的鉴定”J Human Genet。

Xin,Z.: "A novel imprinted gene, KCNQ1DN, within the WT2 critical region of human chromosome 11p15.5 and its reduced expression in Wilms' Tumors."J.Biochem.. 128. 847-853 (2000)

Xin, Z.：“人类染色体 11p15.5 WT2 关键区域内的一种新型印记基因 KCNQ1DN 及其在肾母细胞瘤中的表达减少。”J.Biochem.. 128. 847-853 (2000)

Zhu X.: "C11orf21, a novel gene within the Beckwith-Wiedemann syndrime region in human chromosome 11p15.5"Gene. 256. 311-317 (2000)

朱X.：“C11orf21，人类染色体11p15.5 Beckwith-Wiedemann综合征区域内的一个新基因”基因。