A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells.
A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells.
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一种新的附加报告酶，甲藻荧光素酶，用于监测哺乳动物细胞中的基因表达。
DOI：
10.1016/j.gene.2004.09.028
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发表时间：
2005
期刊：
影响因子：
3.5
通讯作者：
Y. Ohmiya
中科院分区：
文献类型：
--
作者：
C. Suzuki;Y. Nakajima;H. Akimoto;Chun Wu;Y. Ohmiya
甲藻荧光素酶（DL）催化甲藻荧光素被分子氧氧化，产生发射蓝光（λmax=474 nm）的电子激发物种。荧光素酶在其单一多肽链中具有三个催化结构域（Mr=约1.5）。140 kDa），并且每个结构域（约40 kDa）在重组融合蛋白中在E.杆菌因此，DL应该是有用的报告酶在哺乳动物细胞中的基因表达的研究。将由荧光素酶cDNA的一个结构域（dDL）连接到不同的几个启动子组成的表达质粒导入一系列哺乳动物细胞系中。转染后，细胞提取物中的dDL活性通过荧光素酶活性的快速发光测定来测定。对于双重和多重报告基因测定，可以将dDL交换为萤火虫或海肾荧光素酶，并将新的荧光素酶用于对照或靶报告基因。因此，三重报告基因分析可以同时鉴定不同基因的三种转录活性。这项工作建立了DL基因作为一种新的有效的标记基因表达的哺乳动物细胞。
Dinoflagellate luciferase (DL) catalyses the oxidation of dinoflagellate luciferin by molecular oxygen, resulting in an electronically excited species that emits blue light (λmax=474 nm). Luciferase has three catalytic domains in its single polypeptide chain (Mr=ca. 140 kDa), and each domain (about 40 kDa) is enzymatically active when expressed individually in recombinant fusion proteins in E. coli. Thus, DL should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of one domain of luciferase (dDL) cDNA linked to different several promoters were introduced into a series of mammalian cell lines. Following transfection, dDL activities in cell extracts were determined by a rapid light emission assay of luciferase activity. For dual and multiple reporter assays, it is possible to exchange dDL for the firefly or renilla luciferases, and use the new luciferase for control or target reporter genes. Thus, the triple-reporter assay can identify three transcriptional activities of different genes at the same time. This work establishes the DL gene as a new efficient marker of gene expression in mammalian cells.
影响因子：
2.9
作者：
J. Dunlap;J. W. Hastings
通讯作者：
J. Dunlap;J. W. Hastings
DOI：
10.1073/pnas.95.10.5474
发表时间：
1998-05-12
影响因子：
11.1
作者：
Hogenesch, JB;Gu, YZ;Bradfield, CA
通讯作者：
Bradfield, CA