Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector und...Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector under the control of T7 promoter, named pPDGFR-β. Two ribozymes were designed to cleavethe CUU sequence at codon 45 and codon 252 of PDGF receptor β subunit mRNA respectively. These 2 ham-merhead ribozyme genes were cloned into vector PI. 5 between 5' -cis ribozyme and 3' -cis ribozyme to gener-ate the plasmids of pRZ1 and pRZ2. The pPDGFR-β, pRZl and pRZ2 were linearized and then transcribedwith T7 promoter in vitro. Results: The RZ1 showed high cleavage activity in vitro, but the RZ2 showed nocleavage activity under the same condition. Conclusion: The cleavage site selection is an important factor in-fluencing the cleavage activities of ribozymes.展开更多
To investigate the effect of two deoxyribozymes targeting period1(per1)mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases,the specific deoxyribozymes targeting per1 were designed...To investigate the effect of two deoxyribozymes targeting period1(per1)mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases,the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure.per1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1_(164:256).The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions.With the transfection tech-nique mediated by LipofectAMINE^(TM),pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells.The effects of deoxyribozymes on per1 were studied by reverse tran-script-polymerase chain reaction(RT-PCR)and flow cytometry(FCM).When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37℃for 2 h,about 63%of per1_(164:256)RNA transcripts were cleaved by DRz164 and about 50.5%by DRz256.After co-transfecting pcDNA3-per1 with DRz164 or DRz256,the expression of per1 mRNA was de-creased,as indicated by RT-PCR semi-quantity analysis.FCM analysis showed that Per1 protein was inhibited.Both DRz164 and DRz256 targeting per1 have the specific cleavage activity to-ward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.展开更多
基金Supported by the National Natural Science Foundation of China (No. 39870303)
文摘Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector under the control of T7 promoter, named pPDGFR-β. Two ribozymes were designed to cleavethe CUU sequence at codon 45 and codon 252 of PDGF receptor β subunit mRNA respectively. These 2 ham-merhead ribozyme genes were cloned into vector PI. 5 between 5' -cis ribozyme and 3' -cis ribozyme to gener-ate the plasmids of pRZ1 and pRZ2. The pPDGFR-β, pRZl and pRZ2 were linearized and then transcribedwith T7 promoter in vitro. Results: The RZ1 showed high cleavage activity in vitro, but the RZ2 showed nocleavage activity under the same condition. Conclusion: The cleavage site selection is an important factor in-fluencing the cleavage activities of ribozymes.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.3007027&39970275)
文摘To investigate the effect of two deoxyribozymes targeting period1(per1)mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases,the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure.per1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1_(164:256).The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions.With the transfection tech-nique mediated by LipofectAMINE^(TM),pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells.The effects of deoxyribozymes on per1 were studied by reverse tran-script-polymerase chain reaction(RT-PCR)and flow cytometry(FCM).When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37℃for 2 h,about 63%of per1_(164:256)RNA transcripts were cleaved by DRz164 and about 50.5%by DRz256.After co-transfecting pcDNA3-per1 with DRz164 or DRz256,the expression of per1 mRNA was de-creased,as indicated by RT-PCR semi-quantity analysis.FCM analysis showed that Per1 protein was inhibited.Both DRz164 and DRz256 targeting per1 have the specific cleavage activity to-ward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.
