|Budget Amount *help
¥4,100,000 (Direct Cost : ¥4,100,000)
Fiscal Year 2003 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 2002 : ¥3,500,000 (Direct Cost : ¥3,500,000)
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.