Department of Medicine, University of California, Los Angeles, CA, USA.
J Gene Med. 2010 Jun;12(6):528-37. doi: 10.1002/jgm.1463.
Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal.
With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme-substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals.
In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination.
Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.
生物发光成像是一种非侵入性的技术，可定量和定位基因转导和表达。然而，组织在光学区域对光的衰减趋势限制了 BLI 的灵敏度。生物发光报告系统的光输出改进将允许检测到更低水平的表达、更少数量的细胞以及来自动物体内更深和更衰减的组织的表达。
为了确定可提高海肾荧光素酶（RLuc）和高斯荧光素酶（GLuc）报告基因灵敏度的底物，我们评估了天然腔肠素及其三种最有前途的衍生物在转导逆转录病毒载体的培养细胞的 BLI 中的应用。在测试的 8 种酶-底物对中，表现最好的两种进一步在小鼠中进行了评估，以比较它们在活体动物中成像载体修饰细胞的效果。
在细胞培养中，我们观察到各种酶-底物组合的发光水平存在显著差异，发现两种荧光素酶在与底物产生光方面表现出明显不同的能力。最有效的对是 RLuc 与合成腔肠素衍生物 Viviren，以及 GLuc 与天然腔肠素。在动物体内，这两种对的检测灵敏度相似，比原型 RLuc-天然腔肠素组合高 8 到 15 倍。
底物选择可以显著影响 RLuc 和 GLuc 的检测灵敏度，适当选择底物可以大大提高这些酶编码报告基因用于通过 BLI 监测基因转移的性能。
