Document Detail


Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system.
MedLine Citation:
PMID:  18499293     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductions were carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.
Authors:
B A Jardin; Y Zhao; M Selvaraj; J Montes; R Tran; S Prakash; C B Elias
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Publication Detail:
Type:  Journal Article     Date:  2008-05-21
Journal Detail:
Title:  Journal of biotechnology     Volume:  135     ISSN:  0168-1656     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-06-23     Completed Date:  2008-10-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  272-80     Citation Subset:  IM    
Affiliation:
Biotchnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada.
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / metabolism*
Analysis of Variance
Animals
Baculoviridae / metabolism*
Bioreactors*
CHO Cells
Cell Line
Cricetinae
Cricetulus
Filtration
Genetic Vectors
Humans
Insects
Transduction, Genetic*
Chemical
Reg. No./Substance:
EC 3.1.3.1/Alkaline Phosphatase

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


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