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Microinjection of foreign DNA into ascidian embryos

将外源 DNA 显微注射到海鞘胚胎中

基本信息

批准号：
02640571
负责人：
SAIGA Hidetoshi
金额：
$ 1.22万
依托单位：
University of Occupational and Environmental Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02640571/
关键词：
Ascidian Embryo Gene Microinjection Actin 初期発生

项目摘要

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actin gene and that they are classified into at least 4 groups according to the temporal pattern of expression (Saiga, unpublished).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

本研究旨在为建立海鞘胚胎显微注射技术做一些基础性的实验。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）。我选择了“肌动蛋白基因”作为DNA导入海鞘胚胎，因为我发现海鞘具有与鸡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，它们被分为至少4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，可以作为本项目的合适模型系统。我已经分离并鉴定了两个肌动蛋白基因（Saiga，准备中）。一种在母体中存在和/或组成性表达，另一种在64细胞期开始表达。此外，我与京都大学的Satoh博士合作报道了肌肉型肌动蛋白基因的分离和表征（Kusakabe et al.，1991年）。显微注射试验使用与将DNA显微注射到小鼠卵中的装置类似的装置进行，通过将注射针穿过绒毛膜的一侧插入海鞘胚胎中，而另一侧由保持移液管牢固地固定。初步实验表明，注射缓冲液的胚胎中约有一半与对照胚胎一样发育到神经胚。然而，两个冬天的时间还不足以让我熟练掌握这项技术，而且试验次数还不足以建立该技术。将来，我想证明海鞘肌动蛋白基因的启动子/增强子区域可以通过该技术来定义。