尼罗罗非鱼整胚原位杂交技术的建立和初步应用

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

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。

To investigate the expression pattern and function of genes expressed in the development and organogenesis of nile tilapia embryos,whole mount in situ hybridization(WISH) of nile tilapia embryo was established based on the commonly used protocols in zebrafish(Danio rerio) and striped bass(Morone saxatilis).However,the WISH protocols of zebrafish and striped bass are not completely viable for nile tilapia embryos because of their huge,opaque yolk with early pigment formation.Thus we optimized the protocol for its application in nile tilapia.Specifically,we improved the methods of removing the pigments by H2O2 and KOH,and enhanced tissue permeabilization in tilapia embryos using cold acetone instead of proteinase K,reduced the times of washing embryos after being incubated in probes or antibodies,and optimized the system of image collection and the storage of embryos post-WISH.Using the recombination activating gene 1(Rag1) of nile tilapia as the probe gene,the result of WISH in nile 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 nile tilapia was effective and entirely feasible.