Comparison of long-term transgene expression after non-viral and adenoviral gene transfer into primary articular chondrocytes.
Different gene transfer approaches to achieve long-term transgene expression in cultured primary bovine chondrocytes were compared using enhanced green fluorescent protein (EGFP) as a reporter. Transduction with a high-capacity adenoviral vector was 82% efficient when analysed by fluorescence microscopy, while up to 42% of plasmid-transfected cells were EGFP positive with FuGene as a transfection reagent. Rapid dominant marker selection of plasmid-transfected cells was achieved in monolayer culture. With either method of gene transfer, a high proportion of the chondrocytes remained transgene positive during prolonged alginate culture. Transgene transcription in single cells was quantified with a confocal laser scanning microscope. Detection of EGFP expression was more sensitive with this method, identifying more transgene-expressing cells than conventional fluorescence microscopy. Long-term EGFP expression was higher in adenovirally transduced chondrocytes embedded in alginate as compared to plasmid-transfected cells cultured in monolayer or in alginate. Both the adenoviral and the plasmid-based approach appear suited for studies of the molecular and cellular mechanisms by which mutations in cartilage matrix proteins cause disease.
Dinser
R
R
Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany. robert.dinser@med-rz.uni-saarland.de
Kreppel
F
F
Zaucke
F
F
Blank
C
C
Paulsson
M
M
Kochanek
S
S
Maurer
P
P
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002417:Cattle; D019902:Chondrocytes / Q000166:cytology; D004352:Drug Resistance, Microbial; D015870:Gene Expression / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D018613:Microscopy, Confocal / Q000295:instrumentation; D010957:Plasmids / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins