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

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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.