| Blocking caspase-3 activity with a U6 SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta. | |
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MedLine Citation:
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PMID: 14705008 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Apoptosis responding to various insults in bioreactors was observed to reduce cell viability and prevent cells from growing to high density. Inhibition of apoptosis in different ways has proved to be effective in keeping cells viable in high density and result in higher recombinant protein production. In apoptosis, death signals activate a family of proteinases, namely caspases, in a cascade and ultimately activate the final effector proteinase, caspase-3, which cleaves various substrates and drives the cells irreversibly to death. Caspase-3 is the executioner of an apoptotic cell and thus plays a central role in apoptosis. Therefore inhibition of caspase-3 may provide an effective way for apoptosis prevention. In this study, we designed a ribozyme targeted at the 451 nt of hamster caspase-3's open reading frame (ORF) and the ribozyme was proved to be effective in cleaving caspase-3 mRNA in vitro. Then, the ribozyme was cloned into a vector under the control of U6 snRNA promoter, an RNA polymerase III promoter, for high rate of transcription in vivo. The vector was transfected into an interferon-beta producing recombinant CHO cell line, and some clones were screened out that exhibited low caspase-3 production by Western blot analysis. One such clone was then further analyzed and it showed good anti-apoptosis property with respect to cell viability, cell density, and interferon-beta production. |
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Authors:
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Dazhi Lai; Ling Fu; Shaojie Weng; Lianquan Qi; Changming Yu; Ting Yu; Haitao Wang; Wei Chen |
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Publication Detail:
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Type: Comparative Study; Journal Article |
Journal Detail:
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Title: Biotechnology and bioengineering Volume: 85 ISSN: 0006-3592 ISO Abbreviation: Biotechnol. Bioeng. Publication Date: 2004 Jan |
Date Detail:
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Created Date: 2004-01-05 Completed Date: 2004-08-03 Revised Date: 2008-11-21 |
Medline Journal Info:
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Nlm Unique ID: 7502021 Medline TA: Biotechnol Bioeng Country: United States |
Other Details:
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Languages: eng Pagination: 20-8 Citation Subset: IM |
Copyright Information:
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Copyright 2003 Wiley Periodicals, Inc. |
Affiliation:
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Department of Applied Molecular Biology, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie, Fengtai, Beijing 100071, China. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Antineoplastic Agents / metabolism Apoptosis / physiology* CHO Cells Caspase 3 Caspases / genetics*, metabolism* Cell Survival / physiology Cricetinae Cricetulus Culture Media, Serum-Free Enzyme Activation / genetics Enzyme Inhibitors / metabolism Genetic Enhancement / methods* Interferon-beta / biosynthesis*, genetics Promoter Regions, Genetic Protein Engineering / methods RNA, Catalytic / genetics, metabolism* Recombinant Proteins / biosynthesis Ribonucleoprotein, U4-U6 Small Nuclear / genetics, metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Antineoplastic Agents; 0/Culture Media, Serum-Free; 0/Enzyme Inhibitors; 0/RNA, Catalytic; 0/Recombinant Proteins; 0/Ribonucleoprotein, U4-U6 Small Nuclear; 77238-31-4/Interferon-beta; EC 3.4.22.-/Caspase 3; EC 3.4.22.-/Caspases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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