Smale Stephen T
Cold Spring Harb Protoc. 2010 May;2010(5):pdb.prot5423. doi: 10.1101/pdb.prot5423.
When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. Although the Escherichia coli lacZ gene, encoding beta-galactosidase (beta-gal), can be used as a standard reporter for monitoring the strength of a promoter or enhancer in a transient or stable transfection assay, it is predominantly used as an internal control during transient transfection experiments. When used in this manner, cells are usually transfected with the control plasmid (containing a ubiquitously active viral promoter fused to the E. coli lacZ gene) and an experimental plasmid containing another reporter gene (e.g., luciferase or chloramphenicol acetyltransferase [CAT]) under the control of the promoter or enhancer of interest. The basic colorimetric assay described here is the simplest and least expensive assay for quantifying beta-gal activity. The cells are lysed and, after determining the total protein concentration in the extracts, an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-beta-D-galactopyranoside (ONPG), in a buffer containing sodium phosphate and magnesium chloride. When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.
当为某个启动子开发瞬时或稳定转染分析方法时，一个主要目标是量化启动子强度。由于此类分析中的转染效率可能较低，启动子通常与异源报告基因融合，这些报告基因编码的酶可通过高灵敏度分析方法进行量化。转染细胞群体中报告蛋白的活性或荧光与稳态mRNA水平大致成正比。虽然编码β-半乳糖苷酶（β-gal）的大肠杆菌lacZ基因可作为标准报告基因，用于在瞬时或稳定转染分析中监测启动子或增强子的强度，但它主要在瞬时转染实验中用作内部对照。以这种方式使用时，细胞通常用对照质粒（含有与大肠杆菌lacZ基因融合的普遍活跃的病毒启动子）和实验质粒转染，实验质粒在感兴趣的启动子或增强子的控制下含有另一个报告基因（例如，荧光素酶或氯霉素乙酰转移酶[CAT]）。这里描述的基本比色分析是量化β-gal活性最简单且最经济的分析方法。细胞被裂解，在测定提取物中的总蛋白浓度后，将一份提取物与反应底物邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）在含有磷酸钠和氯化镁的缓冲液中混合。当黄色产物可见时，用分光光度法测定样品的光密度。
