Microinjection of foreign DNA into ascidian embryos

本研究旨在为建立一种海鞘胚胎显微注射方法进行一些基础性试验。该项目由两个分项目组成；一个是分离海鞘肌动蛋白基因(1)，另一个是开发海鞘胚胎显微注射技术(2)1。我选择将“肌动蛋白基因”作为DNA引入到海鞘胚胎中，因为我发现海鞘中有与鸡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，它们至少被分为4个组(Saiga，未发表)。它们的5‘端区域可能负责它们不同的时间表达模式，应该作为这个项目的合适的模型系统。我已经分离并鉴定了两个肌动蛋白基因(SAIGA，正在准备中)。一种是母系存在和/或结构性表达，另一种是在细胞期开始表达。此外，我还与京都大学的Satoh博士合作，报告了肌型肌动蛋白基因的分离和特征(Kusakabe等人，1991年)。显微注射试验使用了类似于将DNA显微注射到小鼠卵子中的装置，即通过绒毛膜的一侧将注射针插入到海囊胚胎中，而另一侧用固定吸管牢牢固定。初步实验表明，注射缓冲液的胚胎中约有一半发育到神经细胞凝视状态，与对照胚胎相同。然而，两个冬天的时间对我来说并不足以熟练掌握这项技术，试验的数量还不足以建立这项技术。在未来，我想证明可以用这项技术来确定海鞘肌动蛋白基因的启动子/增强子区。

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actin gene and that they are classified into at least 4 groups according to the temporal pattern of expression (Saiga, unpublished).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.