Barnhart K M, Hartikka J, Manthorpe M, Norman J, Hobart P
Department of Molecular Biology, Vical Incorporated, San Diego, CA 92121, USA.
Hum Gene Ther. 1998 Nov 20;9(17):2545-53. doi: 10.1089/hum.1998.9.17-2545.
Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.
在荧光素酶报告基因质粒中，将来自各种肌肉特异性基因的增强子和启动子替换为人巨细胞病毒（CMV）IE基因的增强子/启动子，或与之组合，目的是鉴定在直接肌内注射后具有增强表达活性的新型启动子嵌合体。相对于CMV调控序列，调控序列的替换或添加在内容、位置和方向上各不相同。使用标准小鼠肌内注射试验在体内定量比较衍生物和亲本质粒的表达活性，并通过转染分化的C2C12小鼠成肌细胞和BHK仓鼠肾细胞在体外进行比较，以测试培养细胞转染是否可以替代至少一些动物实验。在体内，19种增强子/启动子嵌合体中有1种提高了表达水平。在体外，一些嵌合体在C2C12细胞中显示出显著的表达增强，但在BHK细胞中没有。我们得出结论，由于质粒表达谱的差异，这些细胞培养系统不能轻易替代新质粒构建体的体内测试。
