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AIM:To prepare hammerhead ribozymes against mousecaspase-7 and identify their cleavage activity in vitro,inorder to select a ribozyme with specific cleavage activityagainst mouse caspase-7 as a potential gene therapy forapoptosis-related diseases.METHODS:Anti-caspase-7 ribozymes targeting sites 333and 394 (named Rz333 and Rz394) were designed bycomputer software,and their DNA sequences encodingribozymes were synthesized.Caspase-7 DNA sequencewas acquired by RT-PCR.Ribozymes and caspase-7 DNAobtained by in vitro transcription were cloned into pBSKneoU6’ and pGFM-T vectors,respectively.The cleavage activityof ribozymes against mouse caspase-7 was identified bycleavage experiments in vitro.RESULTS:Rz333 and Rz394 were designed and their DNAsequences were synthesized respectively.The expressionvector of caspase-7 and plasmids containing Rz333 andRz394 were reconstructed successfully.Ribozymes andcaspase-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 wasmixed with Rz333 in equivalent,and the cleavage efficiencywas 67.98%.No cleaved segment was observed whencaspase-7 mRNA was mixed with Rz394.CONCLUSION:Rz333 can site-specific cleave mousecaspase-7 mRNA,and it shows a potential for gene therapyof apoptosis-related diseases by down-regulating geneexpression of caspase-7.
AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activity in vitro, inorder to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy forapoptosis-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 DNAobtained by in vitro transcription were cloned into pKKneoU6 ’and pGFM -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 andRz394 were reconstructed successfully .Ribozymes andcaspase-7 mRNA were expressed by in vitro transcription.In vitro cleavage experiment showed tha t 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.