EUKARYOTIC TRANSIENT EXPRESSION SYSTEM DEPENDENT ON TRANSCRIPTION FACTORS AND REGULATORY DNA-SEQUENCES OF VACCINIA VIRUS
EUKARYOTIC TRANSIENT EXPRESSION SYSTEM DEPENDENT ON TRANSCRIPTION FACTORS AND REGULATORY DNA-SEQUENCES OF VACCINIA VIRUS
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DOI：
10.1073/pnas.82.1.19
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
COCHRAN, MA;MACKETT, M;MOSS, B
开发了一种瞬时表达系统，其中嵌合基因在感染牛痘病毒的[猴肾]细胞中表达。构建了含有与原核氯霉素乙酰转移酶（CAT）基因编码片段连接的痘苗病毒基因启动子区域的重组质粒。当通过转染将质粒引入牛痘病毒感染的细胞中时，嵌合基因得到表达，并积累了显着水平的CAT。当将相同的重组质粒引入未感染的细胞时，未检测到CAT活性；当质粒中不存在痘苗病毒启动子或被猿猴病毒40或劳斯肉瘤病毒启动子取代时，也未检测到活性。这种特异性表明表达依赖于重组质粒内的顺式作用痘苗病毒启动子区域和病毒感染过程中产生的可扩散的反式作用转录因子。插入痘苗病毒启动子区域上游的猿猴病毒 40 增强子元件缺乏作用，也将该系统与依赖 RNA 聚合酶 II 的系统区分开来。尽管在未感染或痘苗病毒感染的细胞中均未检测到重组质粒的复制，但 DNA 合成抑制剂显着降低了 CAT 表达。病毒DNA模板的复制可以增强重组质粒中嵌合基因的转录。
A transient expression system in which chimeric genes are expressed in [monkey kidney] cells infected with vaccinia virus was developed. Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed. When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated. CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters. This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection. The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II. Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression. Replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.