A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays.
A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays.
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一种基于聚合酶链反应的方法，用于在瞬时转染测定中检测和定量报告基因表达。
DOI：
10.1006/abio.1993.1171
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发表时间：
1993
影响因子：
2.9
通讯作者：
Gottlieb,DI
中科院分区：
文献类型：
--
作者：
Morales,MJ;Gottlieb,DI
在高等真核生物中，转录通常是通过使用瞬时转染实验来研究的。这些实验是通过用报告基因克隆假定的顺式作用转录元件(即启动子或增强子)来进行的，该报告基因编码靶细胞不表达的蛋白质。虽然这种方法在许多情况下是有用的，但报告分析的有限灵敏度可能会阻止在可获得的细胞很少或转染率较低的情况下进行研究。我们提出了另一种方法。将含有人类生长激素(HGH)报告基因启动子的质粒导入细胞。在孵化一段时间后，RNA被分离出来，并产生与生长激素mRNA互补的DNA。用定量聚合酶链式反应(PCR)检测报告基因的浓度。我们设计了跨越人生长激素基因的mRNA剪接点的聚合酶链式反应引物，这确保了hGH基因的独占扩增，而不是报告质粒的扩增。该方法灵敏，操作简单，不需要特殊的设备，可以在很大范围内定量报告基因的浓度。
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.