Objective To study the transcription effects and cleavage activities of rat caspase-3 specific hammerhead ribozyme (Rz107) in both cell-free conditions and BRL-3A cells. Methods Rat caspase-3 gene fragment was clone...Objective To study the transcription effects and cleavage activities of rat caspase-3 specific hammerhead ribozyme (Rz107) in both cell-free conditions and BRL-3A cells. Methods Rat caspase-3 gene fragment was cloned into the pGEM-T EASY vector under the T7 promoter control. The 32 P-labeled caspase-3 transcript was the target-RNA. Rz107 genes designed against caspase-3 mRNA were cloned into vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32 P-labeled ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Rz107 was electroporated into BRL-3A cells and the Rz107 expression was analyzed by RT-PCR. Results In cell-free conditions, Rz107 was active at 37℃. The optimal temperature was 50℃. The Km and kcat were 14.13?nmol/L and 2.31*min-1 respectively. Intracellular cleavage efficiency of Rz107 was 37%, as analyzed by RT-PCR. This indicated that the design of Rz107 was correct, and Rz107 had the activity of common enzymes.Conclusions Rz107 in cell-free conditions possessed perfect specific catalytic cleavage activity, and it can also cleave the target RNA successfully in cells. The results illustrate the feasibility of ribozyme therapy as a potential alternative approach for treating liver disease caused by apoptosis.展开更多
To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ri...To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3 in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.展开更多
基金thegrantsfromtheHighSchoolScience FoundationofShanghai (No 98QN6 0 )andtheChineseAcademyof Sciences (No KSCX2 2 0 4)
文摘Objective To study the transcription effects and cleavage activities of rat caspase-3 specific hammerhead ribozyme (Rz107) in both cell-free conditions and BRL-3A cells. Methods Rat caspase-3 gene fragment was cloned into the pGEM-T EASY vector under the T7 promoter control. The 32 P-labeled caspase-3 transcript was the target-RNA. Rz107 genes designed against caspase-3 mRNA were cloned into vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32 P-labeled ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Rz107 was electroporated into BRL-3A cells and the Rz107 expression was analyzed by RT-PCR. Results In cell-free conditions, Rz107 was active at 37℃. The optimal temperature was 50℃. The Km and kcat were 14.13?nmol/L and 2.31*min-1 respectively. Intracellular cleavage efficiency of Rz107 was 37%, as analyzed by RT-PCR. This indicated that the design of Rz107 was correct, and Rz107 had the activity of common enzymes.Conclusions Rz107 in cell-free conditions possessed perfect specific catalytic cleavage activity, and it can also cleave the target RNA successfully in cells. The results illustrate the feasibility of ribozyme therapy as a potential alternative approach for treating liver disease caused by apoptosis.
基金the High School Science Foundation of Shanghai (Grant No. 98QN60) and the Chinese Academy of Sciences (Grant No. KSCX2-2-04).
文摘To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3 in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.
