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

我们先前报道了一个胃癌家系，该家系具有H-铁蛋白基因中的A49T种系突变（Am J Genet. 69，191 - 197，2001）。因此，本研究通过体外转染H-铁蛋白基因转基因小鼠，分析其A49T突变在胃癌发生中的作用。用脂质体法将A49T突变的H-铁蛋白基因转染COS-1细胞后，细胞内（尤其是细胞核内）8-羟基脱氧鸟苷（8-OHdG）等致突变碱基增多，提示细胞内DNA自发突变和致癌作用。其次，我们构建了A49T突变型H-铁蛋白基因的基因打靶载体，该载体包含剪接受体en-2、脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）、β-半乳糖苷酶、β-geo和SV 40 poly A信号。电穿孔法转染小鼠ES细胞，筛选出68个G418抗性克隆。其中，15个克隆具有适当的同源重组，通过PCR和Southern印迹。在9个克隆中确认了正确的序列。它们被注射到小鼠囊胚中，然后被移植到养父母体内。将嵌合体小鼠与野生型小鼠交配，获得异源小鼠（转基因小鼠）。目前，我们仔细观察转基因小鼠，并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.