Development of knockout mouse of desmoyokin(AHNAK)and analysis of its functio

桥粒肌蛋白(AHNAK)基因敲除小鼠的研制及其功能分析

• 批准号: 11470186
• 负责人: MORI Osamu
• 金额: $ 7.87万
• 依托单位: Kurume University School of Medicine(Department of Dermatology)
• 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-11470186/
• 关键词: Desmosome; Knockoit mouse; Desincykin; AHNAK; Keratinocyte; BS cell; Cell adhesion; molecular biclogy; デスモソーム; 核移行; 細胞膜; デスモヨーキンケラチノサイト; デスモグレイン; エンボプラキン

In the present study, in order to clarify the role of the molecule (desmoyokin/AHNAK protein), we successfully established gene targetting mice (knockout mice) of desmoyokin/AHNAK protein. We screened mouse genomic DNA from mouse genomic DNA library, and prepared a targetting vector for desmoyokin/AHNAK protein. We inserted linealized targetting vector into enbryonic stem cells, and screened homologous recombinated cells. Using the homologous recombinated enbryonic stem cells, we prepare heterozygous mice and finally obtained homozygous knockout mice of desmoyokin/AHNAK protein. We have confirmed by both DNA and protein levels that these targetted mice are really lacking desmoyokin/AHNAK protein. At the same time, we also suceeded in preparation of desmoyokin/AHNAK protein knockout enbryonic stem cells, which should also be useful to examine the role of desmoyokin/AHNAK protein in the cell levels. We will examine the role of desmoyokin/AHNAK protein using these desmoyokin/AHNAK protein knockout mice and knockout enbryonic stem cells. These study should provide us with numerous important insights into the misterious giant protein, desmoyokin/AHNAK protein.

本研究中，为了阐明该分子（desmoyokin/AHNAK蛋白）的作用，我们成功地建立了desmoyokin/AHNAK蛋白基因靶向小鼠（敲除小鼠）。我们从小鼠基因组文库中筛选小鼠基因组DNA，制备了桥粒细胞因子/AHNAK蛋白的靶向载体。我们将线性化的靶向载体插入胚胎干细胞中，筛选同源重组细胞。利用同源重组的胚胎干细胞制备杂合子小鼠，最终获得桥粒细胞因子/AHNAK蛋白纯合子敲除小鼠。我们已经从DNA和蛋白质水平证实，这些靶向小鼠确实缺乏桥粒细胞因子/AHNAK蛋白。同时，我们还成功地制备了desmoyokin/AHNAK蛋白敲除的胚胎干细胞，这也将有助于在细胞水平上研究desmoyokin/AHNAK蛋白的作用。我们将使用这些desmoyokin/AHNAK蛋白敲除小鼠和敲除胚胎干细胞来研究desmoyokin/AHNAK蛋白的作用。这些研究将为我们提供许多重要的见解神秘的巨大蛋白，desmoyokin/AHNAK蛋白。

项目成果

橋本 隆: "皮膚科診療プラクティス"文光堂. 305 (1999)

桥本隆：《皮肤科实践》文科堂 305 (1999)。

Nousari HC et al.: "The mechanism of respiratory failure in paraneoplastic pemphigus"N Eng J Med. 340. 1406-1410 (1999)

Nousari HC 等人：“副肿瘤性天疱疮呼吸衰竭的机制”N Eng J Med。

橋本隆: "「展望」自己免疫性水疱症-臨床でどこまで診断できるか"皮膚病診療. 22. 524-531 (2000)

Takashi Hashimoto：“展望”自身免疫性大疱病 - 临床诊断的程度如何？22. 524-531 (2000)。

Nie Z, Wang N, Amagai M, Hashimoto T: "C-terminus of Desmoyokin/AHNAK protein is mainly located in the nucleus in low calcium condition, but low to normal calcium shift translocates a part of it to cytoplasm"J Invest Dermatol. 114. 1044-1049 (2000)

Nie Z，Wang N，Amagai M，Hashimoto T：“Desmoyokin/AHNAK 蛋白的 C 末端在低钙条件下主要位于细胞核，但低钙到正常钙转移会将其一部分易位到细胞质”J Invest Dermatol。

橋本隆: "皮膚疾患、特に自己免疫性水疱症と自己抗体"日本アフェレシス学会雑誌. 19. 154-160 (2000)

Takashi Hashimoto：“皮肤病，特别是自身免疫性大疱病和自身抗体”，日本血液成分协会杂志 19. 154-160 (2000)。