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 expressio n in cultured primary bovine chondrocytes were compared using enhanced gree n fluorescent protein (EGFP) as a reporter. Transduction with a high-capaci ty adenoviral vector was 82% efficient when analysed by fluorescence micros copy, while up to 42% of plasmid-transfected cells were EGFP positive with FuGene as a transfection reagent. Rapid dominant marker selection of plasmi d-transfected cells was achieved in monolayer culture. With either method o f gene transfer, a high proportion of the chondrocytes remained transgene p ositive during prolonged alginate culture. Transgene transcription in singl e cells was quantified with a confocal laser scanning microscope. Detection of EGFP expression was more sensitive with this method, identifying more t ransgene-expressing cells than conventional fluorescence microscopy. Long-t erm EGFP expression was higher in adenovirally transduced chondrocytes embe dded in alginate as compared to plasmid-transfected cells cultured in monol ayer or in alginate. Both the adenoviral and the plasmid-based approach app ear suited for studies of the molecular and cellular mechanisms by which mu tations in cartilage matrix proteins cause disease.