尼罗罗非鱼整胚原位杂交技术的建立和初步应用

为了研究尼罗罗非鱼胚胎发育和器官形成过程中基因功能和基因表达图式,本研究建立了尼罗罗非鱼的整胚原位杂交流程。尼罗罗非鱼的胚胎具有卵黄大、不透明、色素出现早等特点,因此现有鱼类整胚原位杂交方法不能完全适用于尼罗罗非鱼的胚胎,故本研究做了相应的调整和优化:通过提高H2O2的浓度和添加KOH,改良了尼罗罗非鱼胚胎的色素去除方法;使用冷丙酮代替蛋白酶K在提高胚胎通透性的同时保持胚胎完整;减少了探针回收和抗体回收后的洗涤次数,完善了结果的图像采集和胚胎保存方案。使用尼罗罗非鱼的重组激活基因Rag1作为探针基因,整胚原位杂交结果显示Rag1基因表达的位置与已报道的斑马鱼和日本青鳉的Rag1基因在胚胎中的表达位置高度保守,胚胎完整,基因表达位置清晰可见,表明此套尼罗罗非鱼的整胚原位杂交技术流程成功有效。

A novel protocol of whole mount in situ hybridization(WISH)and its primary application in nile tilapia

To investigate the expression pattern and function of genes expressed in the development and organogenesis of tilapia embryos, whole mount in situ hybridization (WISH) of tilapia embryo was established based on the commonly used protocols in zebrafish (Danio rerio) and striped bass (Morone saxatilis). However, the WISH protocol of zebrafish and striped bass are not completely viable for tilapia embryos because of their huge, opaque yolkwith early pigment formation. Thus we optimized the protocol for its application in tilapia. Specifically, we improved the methods of removing the pigments by H2O2 and KOH, and enhancing tissue permeabilization in tilapia embryos using cold acetone in stead of Proteinase K, reduced the times of washing embryos after incubated in probes or antibodies, and optimized the system of image collection and the storage of embyos post-WISH. Using therecombination activating gene 1 (Rag1) of tilapia as the probe gene, the result of WISH in tilapia embryos showed that its expression pattern was highly conserved compared with zebrafish Rag1 and medaka (Oryzias latipes) Rag1. The embryos post-WISH were unbroken and the gene expression position was distinct. The results indicated that the protocol of WISH in tilapia was effective and entirely feasible.