Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo  

作　　者：徐人欢 刘静 徐丰 江舸 谢青 周霞秋 金由辛 王德宝 

机构地区：[1]DepartmentofInfectiousDiseases,RuijinHospital,ShanghaiSecondMedicalUniversity,Shanghai200025,China [2]StateKeyLaboratoryofMolecularBiology,InstituteofBiochemistryandCellBiology,ShanghaiInstitutesofLifeSciences,ChineseAcademyofSciences,Shanghai200031,China[2]DepartmentofInfectiousDiseases,RuijinHospital,ShanghaiSecondMedicalUniversity,Shanghai200025,China[3]

出　　处：《Science China(Life Sciences)》2001年第6期618-627,共10页中国科学（生命科学英文版）

基　　金：the High School Science Foundation of Shanghai (Grant No. 98QN60) and the Chinese Academy of Sciences (Grant No. KSCX2-2-04).

摘　　要：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.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 cas-pase-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.

关 键 词：RIBOZYME CASPASE-3 TRANSCRIPT cleavage reaction BRL-3A cell. 

分 类 号：R346[医药卫生—基础医学]