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Development of the gene expression inhibition system using ribozyme

使用核酶开发基因表达抑制系统

基本信息

批准号：
14560256
负责人：
KASHIWAZAKI Naomi
金额：
$ 2.62万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

This study inquired paying attention to the ribozyme for the objective of a development of new "gene expression control system" which can control the specific gene expression in mammals specifically on RNA level. In the present study, the mRNA of the enhanced fluorescence protein gene was selected for a target of the ribozyme, because of stable verification, and designed some constructs which express the gene in mammals. These constructs were built so that it might have a CMV-IE promoter in the upstream of the fluorescence protein gene and it might have SV40 terminator down-stream. These constructs were digested by the restriction enzyme to straight chain and the treated DNA, 5 μg/ml, microscopic injected to rat early phase embryo. Consequently, high level of transient expression was observed at 48 and 36 hrs later after exogenously DNA injected in the nuclei of the pronucleus and the 2-cell stage embryos, respectively. Furthermore, for the DNA injection to 2-cell stage embryos, in spite of having introduced DNA into blastomere of one of the two, the phenomenon which fluorescence protein discovers between both these blastomeres was discovered. It was suggested that spermatozoon tail in which this phenomenon is located ranging over both blastomeres involves strongly, and an announcement about these was able to be made positively. Moreover, it succeeded also in production of the transgenics rats which expressed fluorescence protein in the process of these series. On the other hand, when some ribozymes to the mRNA of a fluorescence protein gene were designed and the cleaving efficiency was verified in vitro, the ribozyme succeeded in cleaving target the mRNA at the predicted target site. Therefore, the base to a develop the gene appearance control system of ribozyme was able to be constructed in this research.

本研究探讨了对核酶的关注，目的是开发一种新的“基因表达控制系统”，可以在RNA水平上特异性地控制哺乳动物特定基因的表达。本研究选择荧光蛋白增强基因的mRNA作为核酶的靶标，并设计了一些在哺乳动物中表达该基因的构建体。这些构建使得它可能在荧光蛋白基因的上游有一个CMV-IE启动子，在下游可能有SV40终止子。用限制性内切酶酶切成直链，处理后的DNA 5 μg/ml显微镜注射到大鼠早期胚胎。结果表明，外源DNA分别在原核和2细胞期胚胎细胞核内注入48和36 h后，瞬时表达量较高。此外，将DNA注射到2细胞期胚胎中，尽管已将DNA导入其中一个卵裂球，但发现荧光蛋白在两个卵裂球之间发现了这种现象。提示这种现象所在的精子尾部分布在两个卵裂球上，参与强烈，并且能够积极地宣布这些。在此过程中，还成功地培育出了表达荧光蛋白的转基因大鼠。另一方面，设计了一些针对荧光蛋白基因mRNA的核酶，并在体外验证了其切割效率，核酶成功地在预测的目标位点切割了mRNA。因此，本研究为构建核酶基因外观调控系统奠定了基础。