A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells.

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.

Duke Scholars

Author Dhavalkumar Dhirajlal Patel Medicine, Rheumatology and Immunology

Author Robert Patrick Drucker Pediatrics, Infectious Diseases

Author David James Pickup Molecular Genetics and Microbiology