Microinjection of foreign DNA into ascidian embryos

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actin gene and that they are classified into at least 4 groups according to the temporal pattern of expression (Saiga, unpublished).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