Morales M J, Gottlieb D I
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Anal Biochem. 1993 Apr;210(1):188-94. doi: 10.1006/abio.1993.1171.
Transcription in higher eukaryotes is often studied by the use of transient transfection assays. These experiments are performed by cloning putative cis-acting transcriptional elements (i.e., a promoter or enhancer) with a reporter gene that codes for a protein not expressed by the target cells. Although this approach is useful in many cases, the limited sensitivity of reporter assays can prevent studies in cases where few cells are obtainable or efficiency of transfection is low. We present an alternative approach. Cells are transfected with a plasmid containing a promoter with a human growth hormone (hGH) reporter gene. After an incubation period, RNA is isolated, and DNA complementary to the growth hormone mRNA is produced. The reporter cDNA concentration is measured by quantitative polymerase chain reaction (PCR). We have designed PCR primers that span the mRNA splice sites of the human growth hormone gene; these ensure exclusive amplification of the hGH cDNA and not the reporter plasmid. The assay is sensitive and simple to perform, requires no special equipment, and can quantify reporter cDNA concentration over a broad range.
在高等真核生物中，转录研究通常采用瞬时转染分析方法。这些实验通过将推定的顺式作用转录元件（即启动子或增强子）与编码靶细胞未表达的蛋白质的报告基因进行克隆来进行。尽管这种方法在许多情况下都很有用，但报告基因检测的有限灵敏度可能会妨碍在可获得细胞数量很少或转染效率很低的情况下进行研究。我们提出了一种替代方法。用含有带有人生长激素（hGH）报告基因的启动子的质粒转染细胞。经过一段孵育期后，分离RNA，并产生与生长激素mRNA互补的DNA。通过定量聚合酶链反应（PCR）测量报告基因cDNA浓度。我们设计了跨越人生长激素基因mRNA剪接位点的PCR引物；这些引物可确保仅扩增hGH cDNA而非报告质粒。该检测方法灵敏且操作简单，无需特殊设备，并且能够在很宽的范围内定量报告基因cDNA浓度。
