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Functional analysis of hamartin by use of Tscl knockout mice.

使用 Tscl 敲除小鼠进行 Hamartin 功能分析。

基本信息

批准号：
12680819
负责人：
KOBAYASHI Toshiyuki
金额：
$ 2.11万
依托单位：
Japanese Foundation for Cancer Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680819/
关键词：
Tsc1 gene knockout mouse Tuberous sclerosis Renal tumor Hamartin Animal model 肝血管腫胎生致死

项目摘要

To elucidate the function of tuberous sclerosis 1 (Tsc1) gene product (hamartin) in vivo, we generated a line of Tsci knockout mouse and compared its phenotypes with those of tuberous sclerosis 2 (Tsc2) gene knockout mice. Heterozvgous Tsc1 mutant mice developed renal tumors by 1.5 year of age. Most of these tumors were cyst. adenomas. Hepatic hemangiomas were also developed with high frequency. Some mice developed uterus leiomyosarcomas and tail hemangiomas. In these renal and extra-renalltumors, loss of wild type Tsc1 allele was detected, suggesting that 2-hit of Tsc1 was important step for tumor development. Homozygous Tsc1 mutants died at around embryonic day 10.5 and were frequently associated with neural tube unciosure. These phenotypes were similar to those of Tsc2 knockout mice, suggesting the functional interaction of these two gene products in vivo. However, the development of renal tumors in Tsc1 knockout mice was apparently slower than that of Tsc2 knockout mice. This suggests that mechanism of tumorigenesis associated with Tsc1 mutation may somewhat differ from that associated with Tsc2 mutation. Next, as an in vitro model system for functional analysis of hamartin, we established cell lines from a renal tumor from a Tsc1 knockout mouse. Established cell lines, CACL1s, exhibited epithelial phenotypes and showed the loss of wild-type Tsc1 allele. By western blot analysis using anti-mouse hamartin antibody, loss of hamartin expression was also confirmed. The Erc gene, which is highly expressed in Tsc2-deficient renal tumor cell lines from the Eker rat model, was also highly expressed in CACL1s. CACL1s were not tumorigenic when subcutaneously transplanted into nude mice. Tsc1 knockout mice and hamartin deficient cell lines established in this study will be usefull experimental models for analyses of hamartin function and mechanism of tumorigenesis associated with Tsc1 mutation.

为了阐明结节性硬化症1（Tsc1）基因产物（hamartin）在体内的功能，我们建立了一个Tsci基因敲除小鼠系，并将其表型与结节性硬化症2（Tsc2）基因敲除小鼠的表型进行比较。异质性Tsc1突变小鼠在1.5岁时发生肾肿瘤。肿瘤多为囊肿。腺瘤肝血管瘤也有很高的发生率。部分小鼠出现子宫平滑肌瘤和尾部血管瘤。在这些肾肿瘤和肾外肿瘤中，检测到野生型Tsc 1等位基因的丢失，表明Tsc 1的2次击中是肿瘤发生的重要步骤。纯合子Tsc1突变体在胚胎第10.5天左右死亡，并经常与神经管uncisosure。这些表型与Tsc 2基因敲除小鼠的表型相似，表明这两种基因产物在体内的功能相互作用。然而，Tsc 1基因敲除小鼠肾肿瘤的发展明显慢于Tsc 2基因敲除小鼠。提示Tsc 1突变与Tsc 2突变的肿瘤发生机制可能有所不同。接下来，作为用于Hamartin功能分析的体外模型系统，我们建立了来自Tsc 1敲除小鼠的肾肿瘤的细胞系。建立的细胞系，CACL1s，表现出上皮表型，并显示野生型Tsc1等位基因的损失。通过使用抗小鼠错构蛋白抗体的蛋白质印迹分析，也证实错构蛋白表达的丧失。Erc基因在Eker大鼠模型的Tsc2缺陷型肾肿瘤细胞系中高度表达，在CACL1中也高度表达。CACL1在裸鼠皮下移植后无致瘤性。本研究建立的Tsc 1基因敲除小鼠和hamartin缺陷细胞系为进一步研究hamartin功能和Tsc 1基因突变相关肿瘤发生机制提供了实验模型。