Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Details

Serval ID
serval:BIB_17F90763CED3
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Detection of promoter activity by flow cytometric analysis of GFP reporter expression.
Journal
Nucleic acids research
Author(s)
Ducrest A.L., Amacker M., Lingner J., Nabholz M.
ISSN
1362-4962[electronic]
Publication state
Published
Issued date
2002
Volume
30
Number
14
Pages
e65
Language
english
Notes
Publication types: Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't - Publication Status: ppublish
Abstract
Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.
Keywords
Animals, Antigens, Thy-1, Cell Line, Flow Cytometry, Gene Expression, Green Fluorescent Proteins, Humans, Luciferases, Luminescent Proteins, Mice, Promoter Regions, Genetic, Recombinant Fusion Proteins, Simian virus 40, Transfection
Pubmed
Web of science
Open Access
Yes
Create date
14/03/2008 9:02
Last modification date
20/08/2019 12:48
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