The function of the EAT, an inhibitor of apotptosis, in vivo and its molecular mechanism in disease

细胞凋亡抑制剂EAT的体内功能及其在疾病中的分子机制

• 批准号: 13470053
• 负责人: UMEZAWA Akihiro
• 金额: $ 9.02万
• 依托单位: National Research Institute for Child Health and Development (2002-2004)Keio University (2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470053/
• 关键词: Apoptosis; Development; EAT Gene; ES Cells; Conditional Targetting; Bcl-2 Family; Transgenic Mouse; EAT; bcl; mcl-1; mcl1; トランスジェニック・マウス; ミトコンドリア; 細胞死

EAT/mcl1 gene was isolated as a gene which was up-regulated at the early stage of differentiation of human embryonal carcinoma cells and belonged to the bcl-2 related gene. We have previously shown that it confered resistance to apoptosis induced by chemotherapeutic agents in cultured fibroblasts and mice overexpressing this gene exhibited hyperplasia of pancreatic islet. In order to clarify the function of the EAT/mcl-1 in vivo, we have disrupted the EAT/mcl1 gene in embryonic stem cells and generated knock out mice.We have established 11 embryonic stem cell lines in which one allele of the EAT/mcl1 exon 1 was flanked by loxP sequence and a selection casette. Three lines of mice were generated by injecting the targeted cells into murine early embryos. Crossing with Cre expressing transgenic mice, we have generated heterozygous EAT null mice and floxed mice. This heterozygous mice(EAT-/+) was morphologically indistinguishable with wild type animals. This indicates the half dose of EAT/mcl-1 is sufficient to normal development. Crossing the floxed mice with tissue specific cre expressing mice, we can nullify the EAT gene in conditional manner. We have introduced mox2 cre transgenic mice which express cre in epiblast derived embryonic tissues to knock out the EAT gene.

EAT/mcl 1基因是在人胚胎癌细胞分化早期表达上调的基因，属于bcl-2相关基因。我们以前已经证明，它赋予抵抗化疗药物诱导的细胞凋亡在培养的成纤维细胞和小鼠过表达这个基因表现出胰岛增生。为了阐明EAT/mcl-1基因在体内的功能，我们将EAT/mcl-1基因在胚胎干细胞中进行了敲除，建立了11个EAT/mcl-1基因外显子1的一个等位基因两侧带有loxP序列的胚胎干细胞系，并建立了一个筛选盒。通过将靶细胞注射到小鼠早期胚胎中产生三个品系的小鼠。与表达Cre的转基因小鼠杂交，我们产生了杂合EAT缺失小鼠和floxed小鼠。该杂合子小鼠（EAT-/+）与野生型动物在形态学上无法区分。这表明EAT/mcl-1的一半剂量足以正常发育。将floxed小鼠与组织特异性cre表达小鼠杂交，我们可以条件性地使EAT基因无效。我们已经引入了mox 2 cre转基因小鼠，其在外胚层衍生的胚胎组织中表达cre以敲除EAT基因。

项目成果

Shibata, R: "Correlation between a specific Wilms tumor suppressor gene (Wt1) mutation and the histological findings in Wilms tumor (WT)"J. Med. Genet.. 39・12. E83 (2002)

Shibata, R：“特定 Wilms 肿瘤抑制基因 (Wt1) 突变与 Wilms 肿瘤 (WT) 的组织学发现之间的相关性”J. Med Genet.. 39·12 (2002)。

Suzuki, S.: "Expression of interleukin-6 in cerebral neurons and ovarian cancer tissue in Trousseau syndrome"Clin. Neuropathol.. 21・5. 232-235 (2002)

Suzuki, S.：“Trousseau 综合征中脑神经元和卵巢癌组织中白细胞介素 6 的表达”，Clin. Neuropathol.. 21・5 (2002)。

Imabayashi, H: "Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis."Experimental Cell Research.. 288. 35-50 (2003)

Imabayashi, H：“通过软骨球形成和大规模 cDNA 分析表达谱来实现去分化软骨细胞的再分化和人骨髓基质细胞的软骨形成。”实验细胞研究.. 288. 35-50 (2003)

Ohyama, M.: "Immunologic and histopathologic characterization of active disease model mouse for pemphigus vulgaris"J. Invest. Dermatol.. 118. 199-204 (2002)

Ohyama，M.：“寻常型天疱疮活动性疾病模型小鼠的免疫学和组织病理学特征”J。

Yabe, H.: "Matrix metalloproteinase (MMP)-1 and -3 expression in Ewing's sarcoma may be due to loss of accessibility of the MMP regulatory element to the specific fusion protein in vivo"Biochem. Biophys. Res. Commun.. 293. 61-71 (2002)

Yabe, H.：“尤文氏肉瘤中基质金属蛋白酶 (MMP)-1 和 -3 的表达可能是由于体内 MMP 调节元件与特定融合蛋白的可及性丧失所致”Biochem。