Abstract
DNA double-strand breaks (DSBs) are repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) mechanisms. It has been revealed that NHEJ is the main repair pathway, and gene mutation and deletion are induced in the processes of DSBs repair (DSBR) in mammalian cells. However, these knowledge was mainly obtained by the studies in cultured cells, and the in vivo studies using individual animals are limited. The aim of this study is to establish the procedures to visualize DSBR using GFP as a repair indicator in medaka, Oryzias latipes. Here, we examined the two systems in medaka cultured cells as the first step for in vivo studies in medaka: 1) HR detection system using DR-GFP [Pierce et al, 1999], and 2) gene deletion detection system using Tetracyclin controllable expression vectors [Noda et al, The 49th, 50th Annual Meeting of JRRS]. We propose that it is possible to use both systems and visualize DSBR in medaka cultured cells from comparison of wild type and mutant medaka. By using the HR detection system, it revealed that DSBR via HR pathway was hardly caused in medaka cultured cells in similar way to other mammalian cells. A radiation sensitive mutant medaka cells showed remarkable decrease in HR repair, compared to wild type cells. And we are investigating the DNA sequence of the DSB sites after repair process. On the other hand, the stable cell lines were established, which showed increase in GFP fluorescence depending on the exposed dose using the gene deletion detection system. These systems would allow us to visualize DSBR and gene deletion in vivo by production of transgenic medaka.
