Chen X, Li Y, Xiong K, Xie Y, Aizicovici S, Snodgrass R, Wagner T E, Platika D
Progenitor Inc, Columbus, Ohio 43212-1566, USA.
Cancer Gene Ther. 1995 Dec;2(4):281-9.
We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used. When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues. The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector. Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required.
我们最近开发了一种独特的细胞质瞬时基因表达系统，该系统基于将靶细胞与噬菌体T7 RNA聚合酶（RNAP）和含有T7自基因的质粒DNA载体共转染。由于这个T7系统是自我启动、自我维持的，并且转录不需要细胞因子，因此它很可能在任何哺乳动物细胞中对任何基因都能在体外发挥作用，更重要的是，在体内也能发挥作用。在本研究中，我们证明T7 DNA载体和T7 RNAP可以通过脂质转染有效地共递送至培养细胞。T7系统在包括几种肿瘤细胞系在内的多种哺乳动物细胞中表达不同的靶基因。在一些被T7载体瞬时转染的肿瘤细胞系中，超过30%的细胞能检测到基因表达。无论使用何种细胞系，转染细胞表达的报告酶（荧光素酶）的平均活性相对恒定。当将T7-荧光素酶载体直接注射到小鼠的各种组织中而不使用脂质体时，在注射的肝脏、肌肉、脑和尾部结缔组织中可以发现荧光素酶活性。发现T7系统表达的荧光素酶水平比传统核基因表达载体所达到的水平高200倍，这取决于注射的组织。用T7-β-半乳糖苷酶（β-gal）构建体直接注射肿瘤导致注射部位附近的肿瘤细胞中β-gal基因表达。此外，在小鼠中直接注射T7系统不会产生可检测到的针对T7 RNAP的抗体。这些结果表明，这种基因表达系统可能在许多不同的医学应用中有用，如癌症基因治疗和DNA疫苗接种，这些应用需要瞬时但快速且有效的基因表达。
