Zhou Y, Giordano T J, Durbin R K, McAllister W T
Department of Microbiology and Immunology, Morse Institute of Molecular Genetics, State University of New York, Health Science Center, Brooklyn 11203-2098.
Mol Cell Biol. 1990 Sep;10(9):4529-37. doi: 10.1128/mcb.10.9.4529-4537.1990.
We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.
我们发现，来自脑心肌炎病毒（EMCV）的5'非翻译前导序列可使哺乳动物细胞中由T3 RNA聚合酶合成的转录本以不依赖帽子结构的方式进行翻译。携带T3 RNA聚合酶基因的稳定小鼠细胞系，当氯霉素乙酰转移酶（CAT）基因与EMCV前导序列融合并通过瞬时DNA摄取导入细胞时，在噬菌体启动子的控制下表达CAT基因。此类细胞中的基因表达水平与依赖宿主RNA聚合酶II转录的传统瞬时表达载体所观察到的水平相似或更高。用编码脊髓灰质炎病毒蛋白酶2A蛋白（抑制依赖帽子结构的翻译）的基因共转染细胞，可刺激EMCV-CAT融合基因的表达，这表明EMCV-CAT融合基因是以不依赖帽子结构的方式表达的。将T3 RNA聚合酶基因和EMCV-CAT融合基因导入多种培养的哺乳动物细胞系（HeLa、BSC40、Ltk-、NIH 3T3和C127），表明T3-EMCV表达系统在广泛的细胞类型中均起作用。
