Microinjection of foreign DNA into ascidian embryos

本课题旨在为建立海鞘胚胎显微注射方法进行一些基础试验。该项目包括两个子项目；一个是海鞘肌动蛋白基因的分离(1)，另一个是海鞘胚胎显微注射技术的发展(2)。我选择“肌动蛋白基因”作为DNA导入海鞘胚胎，因为我发现海鞘有与鸡β肌动蛋白基因交叉杂交的基因，并且根据表达的时间模式，它们至少分为4组（Saiga，未发表）。其中可能负责不同时间表达模式的5′区域应作为本项目合适的模型系统。我已经分离并鉴定了两个肌动蛋白基因（Saiga，准备中）。一个是母系存在和/或组成性表达，另一个在64细胞期开始表达。此外，我还报道了与京都大学佐藤博士合作分离和鉴定肌肉型肌动蛋白基因（Kusakabe et al., 1991）。显微注射试验采用了与鼠卵DNA显微注射类似的装置，将注射针通过一侧绒毛膜插入海鞘胚胎，而另一侧则用手持移液管牢牢固定。初步实验表明，注射缓冲液的胚胎约有一半发育为对照胚胎的神经性胚胎。然而，两个冬天的时间不足以让我熟练掌握这项技术，而且试验的次数还不足以建立这项技术。在未来，我想证明启动子/增强子区域的海鞘肌动蛋白基因可以通过这种技术来定义。

In this research project it was intended to carry out some basic trials toward establishment of a microinjection method for ascidian embryos. The project consisted of two subprojects ; one was isolation of ascidian actin genes (1) and the other was development of a technique of microinjection into ascidian embryos (2).1. I chose "actin genes" as DNA to be introduced into ascidian embryos, since I found that ascidians have genes which cross-hybridize to the chicken beta actin gene and that they are classified into at least 4 groups according to the temporal pattern of expression (Saiga, unpublished). The 5' regions of them which are likely to be responsible for their different temporal expression patterns should serve as an appropriate model system for this project. I have isolated and identified two actin genes (Saiga, in preparation). One is present maternally and/or expressed constitutively and the other begins to be expressed at 64 cell stage. In addition, I have reported isolation and characterization of a muscle type actin gene in collaboration with Dr. Satoh, Kyoto University (Kusakabe et al., 1991).2. Trials of microinjection was carried out using a similar setup to that for microinjection of DNA into a mouse egg by putting an injection needle into an ascidian embryo through one side of the chorion while the other side was fixed firmly by a holding-pipette. Preliminary experiments showed that about half of the embryos injected with buffer developed up to neurula stare as the control embryos. However, two winter times turned out to be not enough for me to be skilled in this technique and the number of trials is not yet enough for the technique to be established.In the future, I would like to show that promoter/enhancer regions of the ascidian actin genes can be defined by this technique.