USE OF BACTERIAL AND FIREFLY LUCIFERASES AS REPORTER GENES IN DEAE-DEXTRAN-MEDIATED TRANSFECTION OF MAMMALIAN-CELLS
USE OF BACTERIAL AND FIREFLY LUCIFERASES AS REPORTER GENES IN DEAE-DEXTRAN-MEDIATED TRANSFECTION OF MAMMALIAN-CELLS
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DOI：
10.1016/0003-2697(92)90245-3
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发表时间：
1992-08-01
影响因子：
2.9
通讯作者：
BALDWIN, TO
中科院分区：
文献类型：
--
作者：
PAZZAGLI, M;DEVINE, JH;BALDWIN, TO
本研究的目的是通过将三种不同的荧光素酶基因置于单一报告载体中并在相同的哺乳动物细胞类型中表达它们来比较它们。所研究的荧光素酶基因是来自Photinus pyralis （PP）和Luciola mingrelica （LM）的luc基因，以及来自Vibrio harveyi （VH）的两个细菌荧光素酶亚基的lux AB5基因的翻译融合。本研究还纳入了氯霉素乙酰转移酶(CAT)基因进行比较。用对照品评价相应酶的测定方法的性能，用标定曲线计算转染后表达酶的结果。所有的生物发光检测在批内和批间均具有高重复性（小于15%）。两种检测方法的比较表明，萤火虫荧光素酶对PP和LM的检测灵敏度最高（分别为0.05和0.08 amol），而VH细菌荧光素酶的检测灵敏度分别为5 amol和100 amol。另一方面，转染各种质粒表明，细胞内表达的CAT酶的含量远高于其他荧光素酶基因。VH荧光素酶在哺乳动物细胞中的表达水平非常低，因为哺乳动物细胞生长的温度相对较高，这似乎损害了活性酶的正确折叠。PP和LM荧光素酶均以皮摩尔水平表达，但在转染细胞内，其含量通常比CAT低10至70倍。基于这些结果，在哺乳动物细胞中使用萤火虫荧光素酶作为报告基因，与CAT相比，敏感性的总体提高约为30至50倍。
The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.