Screening of DNA repair deficient Medaka (Oryzias latipes)

我们构建了Medaka光解酶基因的真核表达质粒，并将其导入Medaka细胞中进行体外和体内实验。该表达质粒包含一个巨细胞病毒增强子和一个胸苷激酶启动子，以过表达Medaka的光解酶基因。首先，我们将该构建体转染到培养的Medaka细胞中，并建立了几条转染细胞系。荧光作用下，各转染体对嘧啶二聚体的修复能力增强。在光修复能力增强最明显的转染体中，光解酶基因转录较前体细胞OL32扩增。在该染色剂中，我们还观察到，在400 J/m2紫外线日光灯照射后，经过20分钟的荧光处理，紫外线存活率有所提高。接下来，将表达构建体微注射到单细胞期的Medaka胚胎中。与未处理的胚胎相比，在微注射胚胎中检测到光裂解酶基因的过表达，但我们未能检测到囊胚中期死亡的光反应性显著增加。接下来，我们试图利用不同群体间DNA多态性，利用PCR系统检测Medaka的cpd - photoolyase （phr）基因和p53基因的γ射线诱导缺失突变体。北方种群的雄性接受4.75 Gy γ射线照射，照射后1天至12天与南方种群的雌性交配。我们从伽玛辐照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。我们在异常胚胎中发现了4个phr突变体（313）和1个p53突变体。这一突变率接近先前报道的由SLT系统或AP-PCR系统确定的突变率。连锁标记的缺失表明，在异常发育的胚胎中，伽马射线诱导的缺失区域应大于40 cM。

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.