Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.
Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.
复制标题
DOI：
10.1371/journal.pone.0012441
复制
发表时间：
2010-08-27
期刊：
影响因子：
3.7
通讯作者：
Sayler GS
中科院分区：
文献类型：
--
作者：
Close DM;Patterson SS;Ripp S;Baek SJ;Sanseverino J;Sayler GS
细菌荧光素酶（lux）基因盒由五个基因（luxCDABE）组成，其蛋白质产物协同产生生物发光光信号，排除补充底物添加或外源操作。历史上仅在原核生物中表达，lux操纵子通过多双顺反子、密码子优化的过程重新合成，以首次在体外和体内证明哺乳动物HEK293细胞系中的自我定向生物发光发射。显示自主的体外光产生比与未转染的对照细胞相关的可观察到的背景大12倍。还原核黄素磷酸盐（FMNH 2）的可用性被确定为限制生物发光底物在哺乳动物细胞环境中，即使在添加组成型表达的黄素还原酶基因（frp）从哈氏弧菌。FMNH 2补充导致表达哺乳动物密码子优化的luxCDE和frp基因的细胞中生物发光增加151倍。当皮下注射到裸鼠中时，体内光学成像允许接近瞬时的光检测，其独立地持续60分钟的测定长度，背景可忽略不计。哺乳动物细胞环境中的勒克斯表达的速度、寿命和自给自足性为目前现有的生物发光和荧光成像技术所不能提供的实时目标可视化提供了一种可行且强大的替代方案。
The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.
登录
查看更多内容
影响因子：
3.6
作者：
Contag, CH;Contag, PR;Benaron, DA
通讯作者：
Benaron, DA
DOI：
10.1007/s00259-008-1022-8
发表时间：
2009-05-01
影响因子：
9.1
作者：
Inoue, Yusuke;Kiryu, Shigeru;Ohtomo, Kuni
通讯作者：
Ohtomo, Kuni
DOI：
10.1007/s10295-005-0211-8
发表时间：
2005-03-01
影响因子：
3.4
作者：
Patterson, SS;Dionisi, HM;Sayler, GS
通讯作者：
Sayler, GS
影响因子：
2.9
作者：
DUNN, DK;MICHALISZYN, GA;MEIGHEN, EA
通讯作者：
MEIGHEN, EA
DOI：
10.1016/0006-291x(76)90539-8
发表时间：
1976-01-01
影响因子：
3.1
作者：
MEIGHEN, EA;BOGACKI, IG;MICHALISZYN, GA
通讯作者：
MICHALISZYN, GA