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Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector
G Brightwell1, V Poirier, E Cole
1Department of Pathology and Microbilogy, School of Medical Sciences, University Walk, Bristol, UK.
Gene
|July 18, 1997
Summary
Cytomegalovirus (CMV) expression vectors show cell cycle-dependent gene expression, varying with serum response. Careful interpretation of phenotypic changes in transfected cells is crucial due to this CMV promoter variability.
Area of Science:
- Molecular Biology
- Cell Biology
Background:
- Cytomegalovirus (CMV)-based mammalian expression vectors are common for driving gene expression in cultured cells.
- WT1 tumor suppressor protein expression was investigated using CMV vectors.
Purpose of the Study:
- To investigate the cell cycle dependence and serum responsiveness of gene expression driven by the CMV promoter/enhancer system.
- To assess the variability in recombinant protein expression from CMV vectors.
Main Methods:
- Immunofluorescent staining, Western blot, Fluorescence-activated cell sorting (FACS) analysis, and Northern blot analysis were employed.
- Expression levels of WT1 protein, WT1 cDNAs, and a luciferase reporter gene were analyzed.
Main Results:
- WT1 protein expression peaked in the S phase and was minimal in G0/G1, indicating cell cycle dependence.
- Both RNA and protein expression from CMV-driven constructs showed cell cycle dependency.
- CMV-driven expression was serum-responsive, increasing 10-fold within 2 hours of serum addition.
Conclusions:
- The CMV promoter/enhancer system exhibits variable responses to cell cycle status and serum availability.
- Researchers must exercise caution when interpreting phenotypic alterations in cells transfected with CMV-based expression vectors due to these expression variabilities.