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.

中文翻译：

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非病毒和腺病毒基因转移到原发性软骨细胞后长期转基因表达的比较。

使用增强的绿色荧光蛋白（EGFP）作为报告基因，比较了在培养的原代软骨细胞中实现长期转基因表达的不同基因转移方法。用荧光显微镜分析时，高容量腺病毒载体的转导效率为82％，而使用FuGene作为转染试剂的质粒转染细胞中，高达42％为EGFP阳性。在单层培养中实现了质粒转染细胞的快速显性标记选择。无论采用哪种基因转移方法，在长时间藻酸盐培养过程中，高比例的软骨细胞仍保持转基因阳性。用共聚焦激光扫描显微镜定量单细胞中的转基因转录。用这种方法检测EGFP表达更为灵敏，与传统的荧光显微镜相比，可鉴定出更多的表达转基因的细胞。与在单层或藻酸盐中培养的质粒转染细胞相比，在藻酸盐中包埋的腺病毒转导的软骨细胞中长期EGFP表达更高。腺病毒和基于质粒的方法都似乎适用于研究软骨基质蛋白突变引起疾病的分子和细胞机制。