鲆鲽鱼类维持干细胞多能性相关转录因子基因的表达、示踪及功能研究

Fish is an important research model as well as one of the main protein sources for human beings. For these reasons, it is necessary not only to recover the genetic resources of endangered fish, but also to preserve excellent aquaculture breeds for long term. And it is of great significance to develop effective and stable methods for preserving fish integrative genetic information. Cryopreservation of fish eggs and embryos is hard to achieve due to the big size and large amounts of yolk. Though it is now possible to cryopreserve fish sperm, this alone could not help to preserve or recover the comprehensive genetic resources. Stem cells, which are pluripotent and convenient for cryopreservation, can be applied to genetic engineering, cell transplantation and other kinds of manipulations. In this sense, stem cells are the ideal types for fish genetic resources preservation and cell engineering breeding. In this study, molecular mechanisms of Oct4, Nanog and Sox2 transcriptional factors in maintaining pluripotency of stem cells will be researched. Expression plasmid for specific stem cells will be constructed using the transcriptional factors' promoter sequences, and will be micro-injected to embryos as the label for stem cells to display and track them during the embryonic development. Meanwhile, knockdown and overexpression techniques will be investigated to study the function of the transcriptional factors in maintaining pluripotency of stem cells. In this way, our study will be a great help for excellent fish breeds and endangered fish germplasms preservation, and will lay a foundation for cell engineering breeding in fish.

鱼类是重要的研究模型，也是人类重要的蛋白质来源，濒危鱼类的遗传资源需保护，水产优良品种的种质需长期保存，建立鱼类完整遗传信息长期稳定的保存方法意义重大。但鱼类卵子和胚胎体积大、卵黄多，难以冷冻保存，鱼类精子可以冷冻保存，但单纯精子保存无法实现物种遗传资源的全面保存与恢复。干细胞具有多潜能分化的能力且便于冷冻保存，可以对其进行各种遗传改造并进行细胞移植等操作，是鱼类遗传资源保存和细胞工程育种的理想细胞类型。本课题拟研究维持鲆鲽鱼类干细胞多能性相关的转录因子Oct4、Nanog和Sox2基因的分子基础，利用其启动子序列构建干细胞特异的表达载体，通过显微注射对干细胞进行标记、识别并对其在胚胎发育整个过程中进行示踪，同时利用基因敲除和过量表达技术，探究这些转录因子基因在维持干细胞多能性中的作用，为利用多能性干细胞开展鱼类优良品种和濒危物种的种质保存以及细胞工程育种奠定基础。

本课题以牙鲆为实验对象，研究了维持鲆鲽鱼类干细胞多能性相关的转录因子基因Oct4、Nanog、Sox1、Sox2、Prdm1和Prdm14的分子基础，利用这些基因的启动子序列构建了干细胞特异的表达载体，通过显微注射对干细胞进行了标记、识别并对其在胚胎发育整个过程中进行示踪，同时利用基因敲减和过量表达技术，探究了这些转录因子基因在维持细胞多能性中的作用。该课题所获得的成果将为利用多能性干细胞开展鱼类优良品种和濒危物种的种质保存以及细胞工程育种奠定基础。