Screening Live Organisms for Mutagenicity

描述（由申请人提供）：已构建转基因小鼠、大鼠和鱼类模型，可用于研究化学品对产生突变的影响。在所有这些生物体中，该过程包括用化学品挑战，然后分离DNA并筛选代表可选择细菌标记的转基因突变。虽然这允许一定程度的定量，但所揭示的结果受到处理时间和从中获得DNA的组织的选择的限制。 在这项提案中，我们计划在转基因胚胎和幼虫中开发一种成像检测方法，这将允许对潜在癌症治疗产生的突变进行活的、完整的动物筛选。这种方法将利用斑马鱼的小尺寸，允许在整个胚胎发育过程中以及胚胎发育之后进行显微镜观察。通过评估活的个体器官的发育，可以很容易地评估新的癌症治疗剂的潜在毒性，包括剂量，以及生物体的生化处理是否可能产生由进行处理的器官定位的副产物。 该技术将通过使用转基因技术与荧光报告靶相结合来开发，所述荧光报告靶以未突变的形式将荧光定位于生物体细胞的特定区域，并且如果突变，则将发生信号变化，从而允许扫描整个胚胎的体细胞突变。 具体目标是： 1)开发荧光指示剂报告基因，如果在靶区域发生突变，将导致荧光重新定位到细胞的另一个区域 2)利用DNA显微注射技术和假型逆转录病毒载体感染技术，构建、鉴定和鉴定携带这些指示基因的转基因斑马鱼胚系 3)评价具有特征诱变剂（ENU）的转基因品系，以检验原理证明

DESCRIPTION (provided by applicant):Transgenic mouse, rat and fish models have been constructed which allow the investigation of the effects of chemicals upon producing mutations. In all these organisms the process involves challenge with the chemical followed by isolation of the DNA and screening for mutations in transgenes representing selectable bacterial markers. While this allows for some degree of quantitativeness, the results uncovered are limited by the processing time and the selection of tissue from which DNA is obtained. In this proposal we plan to develop an imaging assay in transgenic embryos and larvae which will allow for a live, whole animal screen for mutations produced by potential cancer therapeutics. This approach will take advantage of the small size of zebrafish, allowing for microscopical observation throughout and beyond embryonic development. By evaluating live individual organs as they develop, the potential toxicity of new cancer therapeutics can be easily evaluated as to dose, and whether biochemical processing by the organism might create a by-product that is localized by the organ that does the processing. This technology will be developed by using transgenic technologies in combination with fluorescent reporter targets which, in unmutated form, will localize fluorescence to specific regions of the cells of the organism and, if mutated, a signal change would occur allowing one to scan the whole embryo for somatic mutations. The specific aims are: 1) to develop fluorescent indicator reporter genes that will, if mutated in a target region, cause fluorescence to re-locate to another region of the cell 2) to construct, identify and characterize germ-line transgenic zebrafish with these indicator genes using DNA microinjection technology and pseudotyped retroviral vector infection technology 3) to evaluate the transgenic lines with a characterized mutagen(ENU) to test the proof of principle