Fluorescent proteins in animal cells for process development: Optimization of sodium butyrate treatment as an example

Autor: Florian M. Wurm, Martin Jordan, Pascal Batard, L. Hunt
Rok vydání: 2002
Předmět:
Luminescent Proteins/*genetics/metabolism
Cell Culture Techniques/*methods
Green Fluorescent Proteins
Cell Culture Techniques
Clone (cell biology)
Gene Expression
Cell Count
Bioengineering
CHO Cells
Butyrate
Biology
Fluorescence/*methods
Applied Microbiology and Biotechnology
Green fluorescent protein
Flow cytometry
Dose-Response Relationship
chemistry.chemical_compound
Cricetinae
medicine
Animals
Glucose/metabolism
Transgenes
Recombinant Proteins/genetics/metabolism
CHO Cells/cytology/physiology
Microscopy
Dose-Response Relationship
Drug
medicine.diagnostic_test
Chinese hamster ovary cell
Sodium butyrate
Recombinant Proteins
Butyrates
Luminescent Proteins
Glucose
Microscopy
Fluorescence
Biochemistry
chemistry
Cell culture
Indicators and Reagents
Drug
Butyrates/*pharmacology
Plate reader
Indicators and Reagents/*metabolism
Biotechnology
Zdroj: Biotechnology and Bioengineering. 77:528-537
ISSN: 1097-0290
0006-3592
DOI: 10.1002/bit.10108
Popis: Fluorescent proteins expressed in mammalian cells can be quantified quickly and noninvasively with a standard fluorescence plate reader. We have previously exploited this quality in cell growth assessment (Hunt et al., 1999b). In this work, different CHO cell lines constitutively expressing fluorescent proteins were evaluated as model systems for process development and optimization. Our results demonstrate that the fluorescence of these cell lines quickly reveals conditions that might improve the overall productivity. Sodium butyrate, a well-known yet unpredictable enhancer of production, was chosen for this study. Due to the competing effects of sodium butyrate ("butyrate") on expression and cell number, the maximal overall productivity represents a compromise between enhancement of production and toxicity. Based on fluorescence only, it is possible to separate effects on cell number and specific production by combining microplate fluorescence measurements with data obtained by flow cytometry. This allows for rapid screening of different clones without counting cells or quantifying the recombinant protein, a highly attractive feature if the expression of green fluorescent protein (GFP) was correlated to that of a protein of interest. For all clones tested, negative effects of butyrate on proliferation were similar, while net enhancement of expression was characteristic for each clone. Therefore, it is necessary to optimize treatment for each individual clone. This work demonstrates that, based on the fluorescence of GFP-expresssing cell lines, it is possible to examine noninvasively three critical, generic parameters of butyrate treatment: butyrate concentration, exposure time, and culture phase at the time of addition.
Databáze: OpenAIRE
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