Nucleic Acids Research, 1992, Vol. 20, No. 9 2233-2239
© 1992
MOLECULAR BIOLOGY |
Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector
PHLS Centre for Applied Microbiology and Research Porton Down, Salisbury, Wiltshire SP4 0JG, UK
Received February 26, 1992. Revised March 25, 1992. Accepted March 25, 1992.
A defective adenovirus (Ad) type 5 E1-vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd3I respectively. In human fibroblasts infected with RAd35 ß-galactosidase (ß-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with ß-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive ß-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV.
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