Functional analysis of hamartin by use of Tscl knockout mice.

为了阐明Tsc1基因产物在体内的功能，我们培育了Tsci基因敲除小鼠，并将其与Tsc2基因敲除小鼠的表型进行了比较。异交Tsc1突变小鼠在1.5岁时发生肾肿瘤。多数为囊肿性肿瘤。腺瘤。肝血管瘤的发生率也很高。部分小鼠出现子宫平滑肌肉瘤和尾部血管瘤。在这些肾脏和肾外肿瘤中，检测到野生型Tsc1等位基因的缺失，表明Tsc1的2-hit是肿瘤发展的重要步骤。纯合子Tsc1突变体在胚胎10.5天左右死亡，通常与神经管不通畅有关。这些表型与Tsc2敲除小鼠的表型相似，提示这两个基因产物在体内的功能相互作用。然而，Tsc1基因敲除小鼠肾肿瘤的发展明显慢于Tsc2基因敲除小鼠。这表明与Tsc1突变相关的肿瘤发生机制可能与与Tsc2突变相关的机制有所不同。接下来，我们从Tsc1敲除小鼠的肾肿瘤中建立细胞系，作为对错构体功能分析的体外模型系统。建立的细胞系CACL1s表现出上皮表型，并显示野生型Tsc1等位基因的缺失。用抗小鼠错构体抗体进行western blot分析，也证实了错构体表达缺失。Erc基因在来自Eker大鼠模型的tsc2缺陷肾肿瘤细胞系中高表达，在CACL1s中也高表达。在裸鼠皮下移植时，CACL1s无致瘤性。本研究建立的Tsc1基因敲除小鼠和错构体缺陷细胞系将为分析与Tsc1突变相关的错构体功能和肿瘤发生机制提供有用的实验模型。

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.