Laser scanning cytometry to quantify gene transfer efficiency and transcriptional activity of EGFP constructs.
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.
Ferrer-Martínez
Andreu
A
Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Catalonia, Spain. aferrer@porthos.bio.ub.es
Gomez-Foix
Anna Maria
AM
D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins