Gussoni E, Wang Y, Fraefel C, Miller R G, Blau H M, Geller A I, Kunkel L M
Division of Genetics, Children's Hospital, Boston, MA 02115, USA.
Nat Biotechnol. 1996 Aug;14(8):1012-6. doi: 10.1038/nbt0896-1012.
To monitor the presence of introduced genes and the distribution of the encoded proteins in host tissues after gene transfer, we combined fluorescence in situ hybridization (FISH) and immunohistochemistry in two separate gene therapy paradigms. In brain tissue sections from animals injected with pHSVlac vector, we localized nuclei containing vector DNA both in cells expressing and not expressing beta-galactosidase (beta-gal). This suggests that the efficiency of gene transfer is affected not only by gene delivery, but also by cellular controls on gene expression. In a second paradigm, following myoblast transplantation, we detected donor nuclei in the muscle of a patient with Duchenne's muscular dystrophy. The donor nuclei were either surrounded by host nuclei or apparently fused in the patient's muscle fiber producing dystrophin. The combined FISH and immunohistochemistry assay offers greater sensitivity and more information than currently used polymerase chain reaction and protein detection methods.
为监测基因转移后导入基因的存在情况以及编码蛋白在宿主组织中的分布，我们在两种不同的基因治疗模式中结合了荧光原位杂交（FISH）和免疫组织化学技术。在注射了pHSVlac载体的动物的脑组织切片中，我们在表达和不表达β-半乳糖苷酶（β-gal）的细胞中都定位到了含有载体DNA的细胞核。这表明基因转移效率不仅受基因递送的影响，还受细胞对基因表达的调控作用影响。在第二种模式中，成肌细胞移植后，我们在一名杜氏肌营养不良症患者的肌肉中检测到了供体细胞核。这些供体细胞核要么被宿主细胞核包围，要么明显在患者的肌纤维中融合并产生抗肌萎缩蛋白。与目前使用的聚合酶链反应和蛋白质检测方法相比，FISH和免疫组织化学联合检测方法具有更高的灵敏度和更多的信息。
