Lentiviral transduction of microglial cells
Lentiviral transduction of microglial cells
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作者：
Balcaitis, S;Weinstein, JR;Möller, T
小胶质细胞是中枢神经系统的常驻免疫细胞。它们的功能类似于组织巨噬细胞，因此，它们与外周巨噬细胞和单核细胞共享许多特性。一个惊人的相似之处是这些细胞很难通过转染或转导进行遗传操作。我们试图克服这一挑战，并产生稳定转导的小胶质细胞系。基于从巨噬细胞获得的令人鼓舞的结果，我们假设慢病毒载体可能在小胶质细胞的转导中提供有价值的工具。使用基于慢病毒的载体系统，在鼠斯坦细胞病毒启动子（MSCV）的控制下表达增强型绿色荧光蛋白（eGFP），我们发现1、10和100 μ g的感染复数（MOI）分别> 70%、> 88%和>95%的细胞。从转导细胞库中，我们建立了具有不同荧光强度的N9和BV-2小胶质细胞系。使用真实的时间-聚合酶链反应（PCR），我们将整合的eGFP拷贝数与通过流式细胞术测量的eGFP荧光相关联。当混合时，可以通过流式细胞术和荧光显微镜分离多达三个具有不同eGFP强度的细胞系。无论是感染还是转基因表达影响小胶质细胞的活化，一氧化氮（NO）的产生，细胞因子的释放，和表面抗原的表达进行评估。我们的发现，小胶质细胞很容易被基于慢病毒的载体转导，这将有助于依赖于遗传操作的研究，并有助于产生转基因细胞系。此外，具有确定的荧光特性的小胶质细胞系的可用性可以取代用于共培养实验中的小胶质细胞鉴定的精细染色程序。(C)2004年威利-利斯。Inc.
Microglial cells are the resident immune cells of the central nervous system. Their function resembles that of tissue macrophages and, as such, they share many properties with both peripheral macrophages and monocytes. One striking similarity is the difficulty with which these cells can be genetically manipulated via transfection or transduction. We have sought to overcome this challenge and generate stably transduced microglial cell lines. Based on encouraging results from macrophages, we hypothesized that lentiviral vectors might provide a valuable tool in the transduction of microglial cells. Using a lentiviral-based vector system expressing enhanced green fluorescent protein (eGFP) under the control of the murine stein cell virus promoter (MSCV), we found that multiplicities of infection (MOIs) of 1, 10, and 100 transduce >70%, >88%, and >95% of the cells, respectively. From the pool of transduced cells, we established lines of N9 and BV-2 microglial cells with distinct fluorescence intensities. Using real time-polymerase chain reaction (PCR), we correlated the integrated eGFP copy numbers to eGFP fluorescence measured by flow cytometry. When mixed, up to three lines with different eGFP intensities could be separated by flow cytometry and fluorescence microscopy. Neither infection nor transgene expression influenced microglial activation as assessed by nitric oxide (NO) production, cytokine release, and surface antigen expression. Our findings that microglial cells are easily transduced by lentiviral based vectors will facilitate research depending on genetic manipulation and help generate transgenic cell lines. In addition, the availability of microglial cell lines with defined fluorescence properties could replace elaborate staining procedures for microglial identification in co-culture experiments. (C) 2004 Wiley-Liss. Inc.