Establishment of the loss-of-function experimental system using the maxizyme in the ascidian embryo.

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It has been very difficult to prove the gene function in ascidians, because the experimental system, which can disturb a certain gene function, has not been established. In this study, two types of the loss-of-function experimental system using ribozymes in the ascidian embryo were intended to develop. One is aimed to abolish zygotically expressed mRNA and to prove the zygotic gene function. The other targets the maternal mRNAs, which are stored in the egg.1 : The plasmid, which carries the human pol III promoter-driven ribozyme sequence, was micro injected into the ascidian fertilized eggs. The eggs were reared until the tailbud stage, and examined for the expression of maxizyme sequence by RT-PCR.Although enough amount to plasmid was retained in the embryo, the maxizyme RNA was not detected. The same plasmid was also introduced into the fertilized egg by electroporation. In this case, only a trace amount of its expression was observed.2 : Active form (DZ) and inactive form (IDZ) of deoxyribozyme were designed, and microinjected into unfertilized eggs. The injected eggs were kept for several hours. Although the DZ efficiently decrease the amount of the target RNA, IDZ had similar and more effect on the target RNA.This showed the antisense effect of ribozymes in the ascidian egg. Moreover, when the DZ- or IDZ-injected eggs was fertilized, the embryos were developed abnormally, even in the condition, at which the target mRNA was not depleted. This suggest the high toxicity of the deoxyribozyme.According to these results, the imperative factors for designing the ribozymes are piled up, and the suitable conditions for introducing the ribozymes was found. Moreover, sensitive RT-PCR method offers a reliable evaluation system, which is indispensable for the establishment of the loss-of-function experiment on the ascidian development.