Bell Peter, Vandenberghe Luk H, Wu Di, Johnston Julie, Limberis Maria, Wilson James M
Gene Therapy Program, Division of Medical Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
J Histochem Cytochem. 2007 Sep;55(9):931-9. doi: 10.1369/jhc.7A7180.2007. Epub 2007 May 17.
We evaluated novel fluorescent proteins (FPs) as reporters for gene transfer in animals and cells with the aim to develop more-sensitive assays for vector development and the optimization of gene transfer strategies in gene therapy. Adeno-associated virus serotype 5 vectors carrying an expression cassette with a chicken beta-actin promoter encoding the green FPs ZsGreen1, AcGFP1, hMGFP (with and without intron), and EGFP and the red FPs DsRed2 and TurboRFP were administered to mice at identical doses for each organ to target liver, lung, and muscle. Despite the fact that all FPs were expressed from an identical vector backbone, the observed number of fluorescent cells and fluorescence intensities varied between, but was consistent within, each combination of a specific protein and organ. The highest number of fluorescent cells was observed in liver with EGFP and in lung with ZsGreen1 and EGFP. In muscle, AcGFP1 and ZsGreen1 produced the most-intense fluorescence in fibers. In contrast, in culture cells, ZsGreen1 showed substantially stronger fluorescence than all other proteins. Our data demonstrate that each FP has tissue-specific expression profiles that need to be taken into consideration when comparing the performance of vectors in different organs.
我们评估了新型荧光蛋白（FPs）作为动物和细胞中基因转移报告基因的性能，目的是开发更灵敏的检测方法，用于载体开发以及基因治疗中基因转移策略的优化。携带表达盒的腺相关病毒5型载体被给予小鼠，该表达盒带有鸡β-肌动蛋白启动子，编码绿色荧光蛋白ZsGreen1、AcGFP1、hMGFP（有内含子和无内含子）、EGFP以及红色荧光蛋白DsRed2和TurboRFP，每个器官给予相同剂量以靶向肝脏、肺和肌肉。尽管所有荧光蛋白均由相同的载体骨架表达，但观察到的荧光细胞数量和荧光强度在每种特定蛋白与器官的组合之间有所不同，但在每种组合内是一致的。在肝脏中观察到EGFP标记的荧光细胞数量最多，在肺中ZsGreen1和EGFP标记的荧光细胞数量最多。在肌肉中，AcGFP1和ZsGreen1在纤维中产生最强的荧光。相比之下，在培养细胞中，ZsGreen1的荧光比所有其他蛋白都要强得多。我们的数据表明，在比较载体在不同器官中的性能时，每种荧光蛋白都有组织特异性的表达谱，需要予以考虑。
