Yang Jianmin, Friedman Michael S, Bian Huimin, Crofford Leslie J, Roessler Blake, McDonagh Kevin T
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0640, USA.
Arthritis Res. 2002;4(3):215-9. doi: 10.1186/ar409. Epub 2002 Feb 28.
We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.
我们正在开发逆转录病毒介导的基因转移技术，将其应用于人成纤维样滑膜细胞（FLS），作为一种表征滑膜细胞病理生理学中涉及的遗传途径的方法。先前的研究表明，基于莫洛尼鼠白血病病毒的载体感染FLS相对困难。为了确定病毒滴度是否影响FLS的转导效率，我们优化了一种快速、高效且廉价的离心方法来浓缩重组逆转录病毒上清液。通过测量转导细胞中病毒增强绿色荧光蛋白转基因的表达，以及分析逆转录病毒上清液中的病毒RNA来评估该技术。通过将病毒上清液离心4小时实现了100倍的浓缩，病毒颗粒回收率达100%。FLS的转导率从不浓缩上清液时的约15%增加到使用浓缩上清液时的近50%。该方案对于需要在原代FLS中实现高效、稳定、高水平转基因表达的研究人员将很有用。
