Abstract:
:Enhanced green fluorescent protein (EGFP) is the preferred reporter protein for real-time detection in individual cells, but its usefulness for gene expression quantification is limited by the sensitivity of standard detection techniques. We tested whether the unique feature of single-cell detection and quantification by laser scanning cytometry permits the evaluation of EGFP gene expression in monolayer cultures of kidney epithelial 293 and C2C12 muscle cells. Cells were transfected with plasmids expressing EGFP under the control of either the cytomegalovirus or muscle promoters: namely, muscle creatine kinase (MCK) and muscle glycogen phosphorylase (MGP). Cell monolayers were laser-scanned, fluorescence-imaged, and recorded. A population of fluorescence-emitting cells was discriminated, their contour area was defined, and the integrated fluorescence was estimated. These data were used to assess gene transfer efficiency in cells transfected with CMV-EGFP, which was higher in 293 than in C2C12 cells. Analysis of fluorescence intensity revealed that, as expected, CMV constructs were highly expressed in both cell types, whereas MCK and MGP constructs showed the highest transcriptional activity in C12C12 cells. In summary, we describe the utility of laser scanning cytometry for the automated estimation of gene transfer efficiency and transcriptional activity of EGFP constructs in cell monolayers.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Ferrer-Martínez A,Gomez-Foix AMdoi
10.2144/02321st01subject
Has Abstractpub_date
2002-01-01 00:00:00pages
62, 64-6issue
1eissn
0736-6205issn
1940-9818journal_volume
32pub_type
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