Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice
Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice
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DOI：
10.1006/mthe.2001.0460
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发表时间：
2001-10-01
影响因子：
12.4
通讯作者：
Gambhir, SS
中科院分区：
文献类型：
--
作者：
Wu, JC;Sundaresan, G;Gambhir, SS
非侵入性监测报告基因表达的能力提供了显着的优势，目前的技术，如死后组织染色或酶活性测定。在这里，我们展示了一种新的方法，重复跟踪萤火虫荧光素酶（FL）在小鼠骨骼肌的体内基因表达的冷却电荷耦合器件（CCD）相机。我们首先表明，冷却的CCD相机提供了一致的和可重复的结果，在+/- 8%的平均值的标准偏差，和检测灵敏度（范围测试：1 × 10（4）- 1 × 10（9）空斑形成单位（pfu））的1 × 10（6）pfu的E1缺失腺病毒表达FL的巨细胞病毒启动子（Ad-CMV-PL）。然后随时间跟踪腺病毒介导的（1 × 10（9）pfu）FL基因表达的持续时间和幅度。免疫活性的瑞士韦伯斯特小鼠的FL基因表达在最初48小时内达到峰值，20天后福尔斯下降98%，并持续> 150天。相比之下，裸小鼠中的FL活性保持升高> 110天。最后，将转导的Swiss韦伯斯特和裸鼠处死，以显示体内CCD信号与体外荧光素酶测定良好相关（r（2）分别= 0.91和0.96）。我们的研究结果表明，冷却的CCD相机的能力，灵敏和非侵入性跟踪FL基因表达的位置，幅度和持久性。在小动物中的基因治疗研究的监测可能会大大有助于进一步扩展这种技术。
The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/- 8% standard deviation from mean values, and a detection sensitivity (range tested: 1 X 10(4) - 1 X 10(9) plaque forming units (pfu)) of 1 X 10(6) pfu of E1 -deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-PL). The duration and magnitude of adenoviral mediated (1 X 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for > 150 days. In contrast, FL activity in nude mice remains elevated for > 110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2) = 0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.