AIM: 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. CONCLUSION: 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.
AIM: To prepare hammerhead ribozymes against mousecaspase-7 and identify their cleavage activity in vitro, in order to select a ribozyme with specific cleavage activity against mousecaspase-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 mousecaspase-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. CONCLUSION: Rz333 can site-specific cleave mousecaspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.
Authors: M Koizumi; Y Ozawa; R Yagi; T Nishigaki; M Kaneko; S Oka; S Kimura; A Iwamoto; Y Komatsu; E Ohtsuka Journal: Nucleosides Nucleotides Date: 1998 Jan-Mar