An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri
An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri
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亚洲文昌鱼未受精卵的高效显微注射方法
DOI：
10.1007/s00427-013-0441-0
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发表时间：
2013-07-01
影响因子：
2.4
通讯作者：
Wang, Yi-Quan
中科院分区：
文献类型：
--
作者：
Liu, Xin;Li, Guang;Wang, Yi-Quan
文昌鱼是研究进化和发育的理想模式动物。然而，作为一种新兴的模式生物，文昌鱼缺乏其他成熟的模式动物所应用的大多数分子技术。显微注射技术是一种强有力的基因操作技术，无疑是研究基因功能和胚胎发育的有效手段之一。虽然该方法已在佛罗里达和欧洲文昌鱼中得到应用，但仍有待优化并引入其他文昌鱼物种。为了将该技术引入本实验室，我们在前人的基础上进行了改进和优化，并成功地对文昌鱼未受精卵进行了显微注射。我们制备了六种用于实践的溶液：200 mM KCl，100 ng/μl actin-LacZ或100 ng/μl actin-RFP载体，无启动子的LacZ或RFP载体和RFP加帽的mRNA。注射KCl的卵受精率达99.2%以上，其中94.3%的卵能正常孵化，55.9%的卵能存活至2日龄幼虫，与正常受精卵相当。注射两种质粒构建体的胚胎也表现出非常高的孵化率和孵化率，但正常发育率略低于KCl注射。在注射胚胎中，91.8%的胚胎表达外源基因LacZ，80.5%的胚胎表达外源基因RFP，它们都是由文昌鱼β-actin基因启动子驱动的。结果表明，本研究成功地建立了文昌鱼未受精卵显微注射技术，提高了文昌鱼显微注射技术的可行性和效率。
Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200 mM KCl, 100 ng/μl actin-LacZ or 100 ng/μl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8 % expressed exogenous gene LacZ and 80.5 % exhibited foreign RFP expression, which were driven by a promoter from amphioxus β-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.