Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo
Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo
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DOI：
10.1016/j.ymthe.2004.10.016
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发表时间：
2005-03-01
影响因子：
12.4
通讯作者：
Breakefield, XO
中科院分区：
文献类型：
--
作者：
Tannous, BA;Kim, DE;Breakefield, XO
光蛋白在促进我们对生物过程的理解方面发挥了重要作用。更广泛的生物相容性，无毒和新颖的报告可以用来扩大这种潜力。在这里，我们描述了从桡足类海洋生物Gaussia princeps的荧光素酶的性质。它是由185个氨基酸（19.9 kDa）组成的单体蛋白，具有短编码序列（555 bp），使其适用于病毒载体。人源化形式的Gaussia荧光素酶（hGLuc）在通过HSV-1扩增子载体递送后在哺乳动物细胞中有效表达。研究发现它无毒且自然分泌，具有与其他腔肠荧光素酶相似的闪光生物发光特征。与在类似条件下表达的萤火虫（hFLuc）和海肾（hRLuc）激酶的人源化形式相比，hGLuc从活细胞及其直接环境产生超过1000倍的生物发光信号强度，并且从单独的活细胞（不包括分泌的荧光素酶）或细胞裂解物产生超过100倍的强度。此外，在标准成像条件下，hGLuc显示出比hRLuc高200倍的信号强度，并且强度与体内hFLuc相当。Gaussia荧光素酶提供了一种灵敏的方法，用于在培养和体内活细胞中成像基因递送和其他事件，具有独特的特征组合，包括高信号强度、分泌和ATP独立性，因此能够真实的实时报告细胞及其环境。
Photoproteins have played a major role in advancing our understanding of biological processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. hGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under standard imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.