Document Detail


Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production.
MedLine Citation:
PMID:  19706449     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.
Authors:
Chia Chu; Vladimir Lugovtsev; Hana Golding; Michael Betenbaugh; Joseph Shiloach
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Intramural     Date:  2009-08-17
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  106     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-10-06     Completed Date:  2009-11-06     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  14802-7     Citation Subset:  IM    
Affiliation:
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Antigens, Viral / metabolism
Cell Adhesion
Cell Line
Cell Proliferation
Dogs
Gene Expression Regulation, Enzymologic
Hemagglutinin Glycoproteins, Influenza Virus / biosynthesis
Humans
Influenza B virus / physiology*
Kinetics
Protein Denaturation
RNA, Messenger / genetics
Sialyltransferases / genetics*,  metabolism
Transgenes*
Virus Replication*
Chemical
Reg. No./Substance:
0/Antigens, Viral; 0/Hemagglutinin Glycoproteins, Influenza Virus; 0/RNA, Messenger; EC 2.4.99.-/Sialyltransferases; EC 2.4.99.3/CMP-N-acetylneuraminate-alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase
Comments/Corrections

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


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