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

对海鞘基因功能的证明一直是非常困难的，因为还没有建立起能够干扰某种基因功能的实验系统。在这项研究中，两种类型的功能丧失实验系统利用核酶在海鞘胚胎中发展。一个目的是消除合子表达的mRNA，并证明合子基因的功能。另一种靶向储存在卵子中的母体mrna。1 .将携带人类pol III启动子驱动核酶序列的质粒微注射到海鞘受精卵中。将卵饲养至尾芽期，用RT-PCR检测最大酶序列的表达。虽然胚胎中保留了足够数量的质粒，但未检测到最大酶RNA。同样的质粒也被电穿孔引入受精卵。在这种情况下，只观察到微量的表达。2：设计脱氧核酶的活性形式（DZ）和非活性形式（IDZ），并微量注射到未受精卵中。注射的卵子保存了几个小时。虽然DZ有效地减少了靶RNA的数量，但IDZ对靶RNA的影响相似且更大。这表明核酶在海鞘卵中具有反义作用。此外，当注射DZ或idz的卵子受精时，胚胎发育异常，即使在目标mRNA未耗尽的情况下也是如此。这表明脱氧核酶具有高毒性。根据这些结果，总结了设计核酶的必要因素，并找到了引入核酶的合适条件。灵敏的RT-PCR方法为海鞘发育功能丧失实验的建立提供了可靠的评价体系。

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.