Development of new assays to evaluate cellular effects of jonizing radiation

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We have created a number of mutant cell lines that lack a specific component of repair system for DNA double strand break (DSB) from hyper-recombinogenic chicken B cell DT40.In this project, we tried to establish several methods to evaluate effects of ionizing radiation on DT40 mutant cells.1.LOH assay. To evaluate LOH, we have created heterozygous and homozygous HPRT mutant cell lines. In chicken, HPRT locus is not on sex chromosomes, so each cell contains two copies of HRPT gene. We determined optimal concentration of 6 TG that kills every wild type cells. At this condition, heterozygous HPRT mutant cells formed colonies at low frequency, however, they stop proliferation and exhibit abnormal morphology. Therefore we could not examine whether they undergo LOH events or non-dysjunction events. Currently we are trying to clone another potential marker gene APRT.2.We developed chicken chromosome painting probe to accurately detect chromosome numbers or translocations.3.Dr Jasin developed a system for evaluation of homology-directed repair of single defined DSB. We applied this system to DT40 by introducing the artificial recombination substrate into ovoalbumin gene using gene targeting. These cells are useful for assaying recombination activity. We are trying to introduce restriction enzyme I-Scel at extremely high efficiency by protein transduction using I-Scel /TAT fusion protein.