A role of mutated H-ferritin gene in famial gastric cancer -Analysis of mechanism using mutated H-ferritin transgenic mouse-

我们之前报道了一个胃癌家系，他们在H-铁蛋白基因(Am J Hum Genet)中有一个A49T的胚系突变。69,191-197,2001)。因此，在本研究中，我们通过对H-铁蛋白基因A49T突变的体外转染和转基因小鼠的分析，探讨该基因突变在胃癌发生中的作用。用脂质体转染法将A49T突变的H-铁蛋白基因导入COS-1细胞后，细胞内(尤其是细胞核内)8-羟基脱氧鸟苷(8-OHdG)等致突变碱基增加，提示DNA自发突变和致癌作用。接下来，我们构建了A49T突变的H-铁蛋白基因打靶载体，包括剪接受体EN-2、脑心肌炎病毒(EMCV)内部核糖体进入位点(IRES)、β-半乳糖苷酶、β-Geo和SV 40聚A信号。用电穿孔法将其导入小鼠ES细胞，筛选出68个G418抗性克隆。通过聚合酶链式反应和Southern杂交，获得了15个同源重组合适的克隆。在9个克隆中证实了正确的序列。他们被注射到小鼠的囊胚中，并被移植到养父母体内。一些嵌合体小鼠出生，并与野生型小鼠交配获得异种(转基因小鼠)。目前，我们仔细观察转基因小鼠，检查它们是否患有胃癌或其他器官的癌症。

We previously reported a family of gastric cancer, who had a germline mutation of A49T in H-ferritin gene (Am J Hum Genet. 69, 191-197, 2001). Therefore, in this study, we investigated the role of A49T mutation in H-ferritin gene in gastric carcinogenesis by in vitro transfection and analysis of transgenic mouse of the gene. When A49T mutated H-ferritin gene was transfected into COS-1 cells by lipofection method, promutagenic bases such as 8-hydroxy-deoxyguanosine (8-OHdG) were increased in the cells (particularly in nucleus), suggesting spontaneous DNA mutagenesis and carcinogenesis. Next, we constructed a gene targeting vector of A49T mutated H-ferritin gene, which included en-2 (splice acceptor), internal ribosome-entry site (IRES) of encephalomyeocarditis virus (EMCV), beta-galactosidase, beta-geo and SV 40 poly A signal. It was transfected into murine ES cells by electroporation, and 68 G418 resistant clones were selected. Of these, 15 clones with appropriate homologous recombination were obtained by PCR and Southern blotting. The correct sequences were confirmed in 9 clones. They were injected into murine blastcysts and they were transplanted into foster parent. Some chimerical mice were born, and heteromice (transgenic mice) were obtained by mating them with wild type mouse. Currently, we carefully observe transgenic mice and examine if they have a gastric cancer or cancers of the other organs or not.