Development of new assays to evaluate cellular effects of jonizing radiation

我们从高度重组的鸡B细胞DT40中创造了许多缺乏DNA双链断裂（DSB）修复系统特定成分的突变细胞系。在本项目中，我们试图建立几种方法来评估电离辐射对DT40突变细胞的影响。LOH化验。为了评估LOH，我们创建了杂合和纯合的HPRT突变细胞系。在鸡中，HRPT基因位点不在性染色体上，因此每个细胞含有两个HRPT基因拷贝。我们确定了6tg杀死所有野生型细胞的最佳浓度。在这种情况下，杂合的HPRT突变细胞在低频率下形成集落，但它们停止增殖并表现出异常形态。因此，我们无法检查它们是否经历了LOH事件或非失联事件。目前我们正在尝试克隆另一个潜在的标记基因APRT.2。我们开发了鸡染色体涂画探针来准确地检测染色体数目或易位。Jasin博士开发了一个系统来评估同源定向修复单一定义的DSB。我们利用基因靶向技术将人工重组底物导入卵白蛋白基因，将该系统应用于DT40。这些细胞可用于测定重组活性。我们正试图通过I-Scel /TAT融合蛋白的蛋白质转导，以极高的效率引入限制性内切酶I-Scel。

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.