Document Detail


RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells.
MedLine Citation:
PMID:  16860584     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Bcl-2 family proteins play a crucial role in the regulation of the mitochondrial pathway that leads to apoptosis. Members of the Bcl-2 family can be divided into the anti-apoptotic proteins such as Bcl-2 and Bcl-X(L), and the pro-apoptotic proteins such as Bax and Bak and the BH3-only proteins. In this study, siRNA constructs to silence the Bax and Bak genes in Chinese hamster ovary (CHO) cells were generated. Stable CHO cell lines in which the expression of Bax and Bak were significantly knocked down were screened by Western blot analysis and confirmed by RT-PCR. CHO cells with both Bax and Bak knocked down showed a clear resistance against cytotoxic lectins and UV irradiation-induced apoptosis. Compared to original CHO-K1 cells, these cells also survived longer when cultured under extreme conditions such as complete nutrient depletion or in high-osmolality medium. CHO cells with both Bax and Bak genes knocked down displayed an extended lifespan as well as higher viable cell densities in fed-batch cultures, both in adherent form on microcarrier beads and in suspension. The IFN-gamma productivity by a rCHO IFN-gamma cell line in which both Bak and Bax were knocked down increased by 35% compared to the control cells. These results indicate that the genetic inactivation of Bax and Bak in recombinant CHO cells can be an effective strategy in delaying the onset of apoptosis in batch and fed-batch cultures.
Authors:
Sing Fee Lim; Kok Hwee Chuan; Sen Liu; Sophia O H Loh; Beatrice Y F Chung; Chin Chew Ong; Zhiwei Song
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Publication Detail:
Type:  Comparative Study; Journal Article     Date:  2006-06-07
Journal Detail:
Title:  Metabolic engineering     Volume:  8     ISSN:  1096-7176     ISO Abbreviation:  Metab. Eng.     Publication Date:  2006 Nov 
Date Detail:
Created Date:  2006-10-18     Completed Date:  2007-01-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9815657     Medline TA:  Metab Eng     Country:  Belgium    
Other Details:
Languages:  eng     Pagination:  509-22     Citation Subset:  IM    
Affiliation:
Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, 06-01 Centros, Singapore 138668, Singapore.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Apoptosis / radiation effects
Blotting, Western
CHO Cells
Cell Culture Techniques
Cell Survival / genetics*
Cloning, Molecular
Cricetinae
Cricetulus
DNA Primers
Molecular Sequence Data
RNA Interference*
RNA, Small Interfering / genetics
Reverse Transcriptase Polymerase Chain Reaction
Ultraviolet Rays
bcl-2 Homologous Antagonist-Killer Protein / genetics,  metabolism*
bcl-2-Associated X Protein / genetics,  metabolism*
Chemical
Reg. No./Substance:
0/DNA Primers; 0/RNA, Small Interfering; 0/bcl-2 Homologous Antagonist-Killer Protein; 0/bcl-2-Associated X Protein

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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