Screening of DNA repair deficient Medaka (Oryzias latipes)

我们构建了Medaka光解酶基因的真核表达载体，并将其导入体外和体内的Medaka细胞。该表达载体含有巨细胞病毒增强子和胸苷激酶启动子，可过表达青竹的光解酶基因。首先，我们将该载体导入培养的Medaka细胞，并建立了多个转染系。在荧光灯作用下，各转染体的嘧啶二聚体修复能力均增强。在光修复能力最强的转染体中，光解酶基因的转录水平较其祖细胞OL32细胞增强。在该转染体中，我们还观察到在400J/m2的紫外线太阳灯照射后，用荧光灯处理20min的紫外线存活率有所提高。接下来，将表达构建物显微注射到Medaka的一个细胞期的胚胎中。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，但我们没有检测到在囊胚中期死亡的光活化能力显著增加。接下来，我们利用不同居群间的DNA多态，尝试用聚合酶链式反应系统检测青竹CPD-光解酶(Phr)基因和p53基因的伽马射线诱导缺失突变体。用4.75Gy射线照射北方种群雄性，照射后1~12d与南方种群雌性交配，得到2182枚正常孵化的胚胎和313枚发育异常的精子或精子细胞。我们在313个异常胚胎中发现了4个phr突变OUR和1个P53突变。这一突变率接近以前报道的由SLT系统或AP-PCR系统确定的突变率。连锁标记的缺失表明，在发育异常的胚胎中，伽马射线诱导的缺失区应大于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.