Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system
Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system
复制标题
DOI：
10.1016/s0006-291x(02)00601-0
复制
发表时间：
2002-06-28
影响因子：
3.1
通讯作者：
Strzadala, L
中科院分区：
文献类型：
--
作者：
Matuszyk, J;Ziolo, E;Strzadala, L
在转录分析中，表达Renilla荧光素酶的质粒（更具体地说是pRL-TK）通常被用作转染效率的内部控制。将实验报告基因转录归一化为内部控制报告基因转录，可以最大限度地减少因转染细胞不同样本之间转染效率差异而导致的结果差异。显然，与其他质粒共转染或应用处理不应影响对照报告基因的活性。在这里，我们报道了pRL-TK质粒编码的对照鼠兔荧光素酶通过与表达孤儿核受体Nur77家族（Nur77, Nurr1, Nor-1）的载体共转染而表达增强，导致检测结果被误解。此外，我们还发现，对于Nurr1， phRG-B（一种含合成Renilla荧光素酶基因的无启动子报告质粒）是比含有HSV-TK的载体更好的对照报告载体。最后，我们注意到Nurr1蛋白对Fas配体启动子驱动的转录缺乏影响。(C) 2002 Elsevier Science （USA）。版权所有。
In transcription assays, Renilla luciferase-expressing plasmids (more specifically pRL-TK) are commonly used as an internal control of transfection efficiency. Normalization of the experimental reporter gene transcription to the internal control reporter gene transcription minimizes variability of obtained results caused by differences in transfection efficiency between different samples of transfected cells. It is obvious that co-transfection with other plasmids or applied treatments should not affect the activity of the control reporter. Here we report that expression of the control Renilla luciferase encoded by pRL-TK plasmid was enhanced by co-transfection with vectors expressing orphan nuclear receptors Nur77 family (Nur77, Nurr1, Nor-1), leading to misinterpretation of the assay results. Further, we show that for Nurr1, phRG-B (a promoterless reporter plasmid containing synthetic Renilla luciferase gene) is a better control reporter vector than HSV-TK containing vectors. Finally, we noted the lack of effect of Nurr1 protein on the Fas Ligand promoter-driven transcription. (C) 2002 Elsevier Science (USA). All rights reserved.