Osorio Johan S, Bionaz Massimo
Department of Animal and Rangeland Sciences, Oregon State University, Corvallis 97331, USA.
Department of Animal and Rangeland Sciences, Oregon State University, Corvallis 97331, USA.
Gene. 2017 Aug 30;626:200-208. doi: 10.1016/j.gene.2017.05.025. Epub 2017 May 10.
Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor β/σ (PPARβ/σ) agonist. The GRT assay was unaffected by poor transfection in MACT cells although the high transfection hampered the possibility of detecting differences between 10 and 100nM of the PPARβ/δ agonist. In MDBK cells, low transfection efficiency (<2.0%) failed to detect any differences with GRT assay. The level of transfection was positively associated with a lower coefficient of variation of GRT data. Overall, our data indicates that results of GRT assays are affected by transfection efficiency and a minimum transfection of 2% is required. Thus, factors such as TR type, TR amount, and DNA plasmid amount need to be optimized for a specific cell type before performing GRT assays.
基因报告技术（GRT）为监测生物事件开辟了几条新途径，包括转录因子的激活，而转录因子的激活是营养基因组学研究的核心。然而，这项技术严重依赖于将外源质粒DNA插入细胞核（即转染），这在细胞类型之间可能极具挑战性且差异很大。本研究的目的是探讨在牛永生化细胞系、马-达二氏牛肾（MDBK）细胞和牛乳腺上皮肺泡（MACT）细胞上生成可靠的GRT检测数据的最佳条件。报告了两个实验的结果。在实验1中，我们使用96孔板和自动倒置荧光显微镜，比较了市售转染试剂（TR）Lipofectamine® 3000（Lipo3）、Lipofectamine® LTX（LipoLTX）和TransIT-X2®（TransX2）、三种剂量的TR（即0.15、0.3和0.4μL/孔）以及三种剂量的绿色荧光蛋白质粒DNA（即10、25和50ng/孔）之间的转染效率。使用CellProfiler软件分析转染效率和死亡率。转染效率在实验结束时（转染后20小时）之前一直在增加，此时MACT细胞的转染率高于MDBK细胞（16.3%对2.2%）。尚不清楚MDBK细胞中转染率低的原因。对于MACT细胞和MDBK细胞，分别使用0.3μL/孔的LipoLTX加25ng/孔的质粒DNA（约29.5±1.9%）和0.15μL/孔的LipoLTX加25ng/孔的质粒DNA（约4.0±0.4%）可获得最大转染效率。TR和DNA用量越高，通常细胞死亡率越高。利用实验1中确定的高、中、低转染效率条件，我们对用10nM或100nM合成过氧化物酶体增殖物激活受体β/σ（PPARβ/σ）激动剂处理的MACT细胞和MDBK细胞进行了过氧化物酶体增殖物激活反应元件（PPRE）荧光素酶的GRT检测。尽管高转染率阻碍了检测10和100nM PPARβ/δ激动剂之间差异的可能性，但GRT检测不受MACT细胞中转染不佳的影响。在MDBK细胞中，低转染效率（<2.0%）未能通过GRT检测发现任何差异。转染水平与GRT数据的较低变异系数呈正相关。总体而言，我们的数据表明GRT检测结果受转染效率影响，且需要至少2%的最低转染率。因此，在进行GRT检测之前，需要针对特定细胞类型优化TR类型、TR用量和DNA质粒用量等因素。
