Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus.
A transient expression system in which chimeric genes are expressed in 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. This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D005057:Eukaryotic Cells / Q000502:physiology; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014615:Vaccinia / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*
D004274:DNA, Recombinant; D004279:DNA, Viral; D014157:Transcription Factors; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase