Development of comprehensive toxicity testing method for polluted water using transgenic zebrafish

在我们开始这项研究之前，我们建立了一个转基因斑马鱼品系，用于检测水中的诱变剂。在该转基因株系中，构建了携带目的基因rpsL(大肠杆菌Sm敏感基因)和救援标记基因kanR(大肠杆菌Km抗性基因)的穿梭载体pML4，并确定了冷冻条件。原液中添加0.1M葡萄糖可提高受精率10-20%。当典型诱变剂MNNG(最大浓度为1 mM)作用于转基因鱼胚胎1h时，突变频率随剂量增加而增加。MNNG引起的主要碱基取代是G：C&gt；A：T，这是典型的烷基化剂引起的转变。10微克/毫升的苯并(A)芘(B(A)P)在代谢激活后诱发突变，经16小时暴露后，这些胚胎均可检测到致突变性。B(A)P的主要碱基替换如预期的那样是G：C&gt；T：A和G：C&gt；C：G。将MNNG暴露于胚胎后，鱼类的身长明显缩短，并在暴露于B(A)P后出现畸形，胚胎形态变化可能也适用于评估化学物质的毒性，我们打算将这种转基因鱼类用于环境监测。将胚胎保存在废物倾倒点溪水中，15%的胚胎出现脊椎水肿或排气，但未检测到致突变性。胚胎形态异常有望成为检测水体污染的良好指标。

Before we started this study, we established a transgenic zebrafish line used for detection of mutagens in water. In this transgenic line, a shuttle vector plasmid, pML4, which carries a target gene, rpsL (Sm sensitive gene of E. coli), and a rescue marker gene, KanR (Km resistant gene of E. coli).For stable maintenance of transgenic line, we determined a condition for freezing the sperms. Addition of 0.1M glucose to a stock solution improved the efficiency of fertilization, which was 10-20%.When MNNG, a typical mutagen, (maximum 1 mM) was exposed to embryos of transgenic fish for 1 hr., mutant frequency was increased dose-dependently. A major base substitution induced by MNNG was G : C > A : T, a typical transition caused by alkylating agent. Mutagenicity of benzo(a)pyrene (B(a)P)., 10 micro-g/ml), which induces mutation after metabolic activation, was detectable by these embryos, by the exposure for 16 hrs. Major base substitution by B(a)P were G : C > T : A and G : C > C : G as expected. By the exposure of MNNG to embryo, length of fish was shortened significantly, and malformation was observed after the exposure of B(a)P. Probably, morphological changing of embryo is also applicable to evaluate the toxicity of chemicals.We intend to use this transgenic fish for environmental monitoring. When the embryos were kept in water collected from the stream from a waste dumping point, 15% of embryos showed edema or vent of vertebra but mutagenicity was not detected. Morphological abnormality on embryo is expected to be a good marker for detecting the pollution of water.