Ray Pritha, Tsien Roger, Gambhir Sanjiv Sam
Molecular Imaging Program at Stanford, Departments of Radiology and Bioengineering, Bio-X Program, School of Medicine, Stanford University, Stanford, California, USA.
Cancer Res. 2007 Apr 1;67(7):3085-93. doi: 10.1158/0008-5472.CAN-06-2402.
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 firefly 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.
使用多种报告基因和成像技术的多模态成像已成为确定小动物体内报告基因表达的位置、强度和时间变化的一项越来越重要的工具。我们已经报道了在细胞培养和活体动物中构建并验证了几个由生物发光、荧光和正电子发射断层扫描（PET）报告基因组成的三重融合基因。然而，与非融合的生物发光和荧光成分相比，这些载体编码的融合报告蛋白的生物发光和荧光成分活性较低。在本研究中，我们首先创建了一个带有过氧化物酶体定位信号的耐热萤火虫荧光素酶（tfl）的突变体（mtfl），使其具有更高的细胞质定位，并改善其底物d - 荧光素的可及性。在细胞培养和活体小鼠中对三种荧光素酶[mtfl、tfl和萤火虫荧光素酶（fl）]进行比较，结果显示mtfl的活性比fl高6至10倍（体外）和2倍（体内）。携带mtfl作为生物发光报告成分的三重融合载体的改进版本显示出比先前的三重融合载体显著更高（P < 0.05）的生物发光。在所评估的三种不同的红色荧光报告基因（分别从水母发色团、珊瑚Heteractis crispa和珊瑚Discosoma分离得到的jred、hcred和mrfp1）中，mrfp1作为三重融合报告基因的一个成分，能够在体内荧光成像中保持最高的表达水平。野生型单纯疱疹病毒1（HSV1）胸苷激酶基因（wttk）的截短版本作为这种改进的三重融合载体的第三个报告成分，其表达水平高于截短的突变体HSV1 - sr39 TK（ttk）。使用生物发光和微型PET对荷瘤小鼠进行多模态成像显示，与表达fl - mrfp1 - wttk的肿瘤相比，表达mtfl - mrfp1 - wttk的肿瘤具有更高的荧光素酶活性[（2.7±0.1对1.9±0.1）×（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（18F - FEAU）摄取水平相似（每克注射剂量的百分比为1.37±0.15对1.37±0.2）。两种肿瘤显示出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生物发光、荧光和微型PET成像技术的组合，对活体动物中数量较少的细胞进行高灵敏度检测。
