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

被引:1
作者
Xu, RH
Liu, J
Xu, F
Jiang, G
Xie, Q
Zhou, XQ [1 ]
Jin, YX
Wang, DB
机构
[1] Shanghai Med Univ 2, Ruijin Hosp, Dept Infect Dis, Shanghai 200025, Peoples R China
[2] Chinese Acad Sci, Shanghai Inst Life Sci, Inst Biochem & Cell Biol, State Key Lab Mol Biol, Shanghai 200031, Peoples R China
来源
SCIENCE IN CHINA SERIES C-LIFE SCIENCES | 2001年 / 44卷 / 06期
关键词
ribozyme; caspase-3; transcript; cleavage reaction; BRL-3A cell;
D O I
10.1007/BF02879356
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
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.
引用
收藏
页码:618 / 627
页数:10
相关论文
共 12 条
[1]  
ANDREW F, 1996, CELL, V85, P781
[2]  
BETTRAND E, 1994, NUCLEIC ACIDS RES, V22, P293
[3]   Proteases to die for [J].
Cryns, V ;
Yuan, JY .
GENES & DEVELOPMENT, 1998, 12 (11) :1551-1570
[4]   Therapeutic applications of ribozymes [J].
Kijima, H ;
Ishida, H ;
Ohkawa, T ;
KashaniSabet, M ;
Scanlon, KJ .
PHARMACOLOGY & THERAPEUTICS, 1995, 68 (02) :247-267
[5]   SELECTION OF EFFICIENT CLEAVAGE SITES IN TARGET RNAS BY USING A RIBOZYME EXPRESSION LIBRARY [J].
LIEBER, A ;
STRAUSS, M .
MOLECULAR AND CELLULAR BIOLOGY, 1995, 15 (01) :540-551
[6]  
LIU J, 2000, HIGH TECHNOLOGY LETT, V6, P84
[7]  
NARENDRA KV, 1998, NUCLEIC ACIDS RES, V26, P5237
[8]   Ribozymes as therapeutic tools for genetic disease [J].
Phylactou, LA ;
Kilpatrick, MW ;
Wood, MJA .
HUMAN MOLECULAR GENETICS, 1998, 7 (10) :1649-1653
[9]   Emerging roles of caspase-3 in apoptosis [J].
Porter, AG ;
Jänicke, RU .
CELL DEATH AND DIFFERENTIATION, 1999, 6 (02) :99-104
[10]  
Sambrook J., 2002, MOL CLONING LAB MANU