Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes.
OBJECTIVE
To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activity in vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases.


METHODS
Anti-caspase-7 ribozymes targeting sites 333 and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained by in vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experiments in vitro.


RESULTS
Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394.


CONCLUSIONS
Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.
Zhang
Wei
W
Department of Infectious Disease, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Xie
Qing
Q
Zhou
Xia-Qiu
XQ
Jiang
Shan
S
Jin
You-Xin
YX
D000818:Animals; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics*; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics
D061945:Caspase Inhibitors; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C505524:Casp7 protein, mouse; D053179:Caspase 7; D020169:Caspases