Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects
Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects
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DOI：
10.1158/0008-5472.can-06-2402
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发表时间：
2007-04-01
期刊：
影响因子：
11.2
通讯作者：
Gambhir, Sanjiv Sam
中科院分区：
文献类型：
--
作者：
Ray, Pritha;Tsien, Roger;Gambhir, Sanjiv Sam
使用几种报告基因和成像技术的多模态成像已经成为确定小动物中报告基因表达的位置、幅度和时间变化的越来越重要的工具。我们已经报道了几个三重融合基因组成的生物发光，荧光，和正电子发射断层扫描（PET）报告基因在细胞培养和活体的建设和验证。然而，由这些载体编码的融合报告蛋白的生物发光和荧光组分具有较低的活动时，与非融合的生物发光和荧光组分相比。在这项研究中，我们首先创建了一个突变体（mtfl）的热稳定性萤火虫荧光素酶（tfl）轴承过氧化物酶体定位信号，具有更大的细胞质定位和改善其底物，D-胰蛋白酶的访问。三种酶之间的比较[mtfl tfl，和萤火虫荧光素酶（FL）]在细胞培养物和活小鼠中均显示MTFL具有6- 10倍的携带mtfl作为生物发光报告组分的三重融合载体的改进形式显示出显著的生物发光活性。（P < 0.05）比先前的三重融合载体更高的生物发光。三种不同的红色荧光报告基因（jred，hcred，和mrfp 1，分别从水母发色团，珊瑚Heteractis crispa，和珊瑚Discosoma，）评估，mrfp 1能够保持最高的表达作为一个组成部分的三重融合报告基因在体内荧光成像。野生型单纯疱疹病毒1型（HSV 1）胸苷激酶基因（wttk）的截短版本保留了比截短突变体HSV 1-sr 39 TK（ttk）更高的表达水平，作为该改进的三重融合载体的第三报告组分。使用生物发光和microPET对荷瘤小鼠进行的多模态成像显示出更高的荧光素酶活性。（2.7 +/- 0.1 vs 1.9 +/- 0.1）X 10（6）p/s/cm（2）/sr），但氟-18-标记的2 '-氟-2'-脱氧阿拉伯呋喃糖基-5-乙基尿嘧啶水平相似与fl-mrfp 1-wttk表达肿瘤相比，mtfl-mrfp 1-wttk表达肿瘤的（18 F-FEAU）摄取[（1.37 +/- 0.15 vs 1.37 +/- 0.2）注射剂量百分比/克]。两种肿瘤均显示18F-FEAU的蓄积比氟-18-标记的9-（4-氟-3-羟甲基丁基）鸟嘌呤高4至5倍（P < 0.05）。这种改进的三重融合报告载体将能够使用组合的生物发光、荧光和microPET成像技术高灵敏度地检测来自活动物的较低数量的细胞。
Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, D-luciferin. Comparison between the three luciferases [mtfl tfl, and flrefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 +/- 0.1 versus 1.9 +/- 0.1) X 10(6) p/s/cm(2)/sr) but similar level of fluorine-18-labeled 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake [(1.37 +/- 0.15 versus 1.37 +/- 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttk-expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques.