BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
Laboratory of Plant Physiology, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
Sensors (Basel). 2018 Feb 16;18(2):602. doi: 10.3390/s18020602.
Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.
在微流控系统中，反向转染的细胞阵列在进行大规模平行筛选 G 蛋白偶联受体 (GPCR) 激活方面具有巨大的潜力。在这里，我们报告了一种使用 HEK293 细胞反向转染、立体荧光显微镜在流室格式下成像、使用荧光蛋白 Cameleon 实时监测细胞质钙离子波动以及分析 GPCR 对连续样品暴露的反应的新型平台的制备。为了确定 DNA 浓度与基因表达之间的关系，我们分析了用编码荧光蛋白的质粒 DNA 的不同浓度制备的细胞阵列和神经激肽 1 (NK1) 受体。我们观察到，无论是特定 DNA 浓度还是总 DNA 浓度，对基因表达都有明显的影响。当用其配体暴露时，含有 NK1 质粒 DNA 的反向转染斑点的总 DNA 浓度为 1%，仍可检测到可察觉的 NK1 激活。通过在反向转染中改变 GPCR DNA 浓度，可以优化受体对连续样品暴露的敏感性和稳健性。显示了一个包含 NK1 受体、苦味受体 TAS2R8 和对照物的阵列的注射系列。两个受体都被两种配体的交替样品暴露了 14 次。特定反应仍然具有可重复性。该平台为 GPCR 文库的高通量筛选带来了新的机会。
