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Screening of DNA repair deficient Medaka (Oryzias latipes)

DNA修复缺陷青鳉（Oryzias latipes）的筛选

基本信息

批准号：
07558288
负责人：
MITANI Hiroshi
金额：
$ 2.69万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558288/
关键词：
Medaka DNA repair UV Photolyase mutation 光回復酸素トランスジェニック PCR トランスジェニック魚類

项目摘要

We constructed a eukaryotic expression plasmid of the Medaka photolyase gene and introduced it into Medaka cells in vitro and in vivo. The expression plasmid contains a cytomegalovirus enhancer and a thymidine kinase promoter to overexpress the photolyase gene of the Medaka. First, we transfected this construct into cultured Medaka cells and established several lines of transfectant. Every transfectant showed enhanced ability of pyrimidine dimer repair in the presence of fluorescent light. In the transfectant that showed the most enhanced ability of photorepair, the augmented transcription of photolyase gene was observed compared with that of progenitor OL32 cells. In this transfectant, we also observed an enhanced rate of UV survival with 20 min of fluorescent light treatment after irradiation with a 400 J/m2 UV sunlamp. Next, the expression construct was microinjected into the embryos of the Medaka at the one cell stage. Compared with the nontreated counterparts, the overexpression of a photo-lyase gene was detected in the microinjected embryos, but we failed to detect a significant increase in photoreactivability of death at the midblastula stage. Next, we tried to detect gamma-ray induced delection mutants of CPD-photolyase (phr) gene and p53 gene of the Medaka by a PCR system utilizing their DNA polymorphism between different populations. Males from the northern population were irradiated with 4.75 Gy gamma-rays and from 1 day to 12 days after irradiation they were mated with females from the southern population.We got 2182 normally hatched embryos and 313 abnormally developed embryos from gamma-irradiated sperm or spermatid. We found 4 phr mutants our of 313 and a p53 mutant among abnormal embryos. This mutation rate is close to previously reported one determined by a SLT system or a AP-PCR system. The loss of linkage markers to suggested that the deletion region induced by gamma-rays should be more than 40 cM in abnormally developed embryos.

我们构建了青鳉光解酶基因的真核表达质粒，并将其体外和体内导入青鳉细胞中。该表达质粒含有巨细胞病毒增强子和胸苷激酶启动子，以过表达青鳉的光裂合酶基因。首先，我们将该构建体转染到培养的青鳉细胞中，并建立了几个转染子系。每个转染子在荧光灯存在下都表现出增强的嘧啶二聚体修复能力。在显示光修复能力最强的转染子中，与祖细胞OL32细胞相比，观察到光裂合酶基因的转录增强。在该转染子中，我们还观察到，在用 400 J/m2 紫外线太阳灯照射后，经过 20 分钟的荧光灯处理，紫外线存活率有所提高。接下来，将表达构建体显微注射到单细胞阶段的青鳉胚胎中。与未处理的胚胎相比，在显微注射的胚胎中检测到光裂解酶基因的过度表达，但我们未能检测到中囊胚阶段死亡光反应性的显着增加。接下来，我们尝试利用不同群体之间的DNA多态性，通过PCR系统检测伽马射线诱导的青鳉CPD-光解酶（phr）基因和p53基因的缺失突变体。来自北方种群的雄性受到4.75 Gy伽马射线的照射，照射后1天到12天，它们与来自南方种群的雌性交配。我们从伽马射线照射的精子或精细胞中获得了2182个正常孵化的胚胎和313个发育异常的胚胎。我们在 313 个异常胚胎中发现了 4 个 phr 突变体和一个 p53 突变体。该突变率接近于之前报道的 SLT 系统或 AP-PCR 系统测定的突变率。连锁标记的丢失表明，在异常发育的胚胎中，伽马射线诱导的缺失区域应该超过40 cM。