Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.
Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use.
Ramage
A D
AD
Division of Molecular Biology, Roslin Institute, Edinburgh, Scotland, UK.
Clark
A J
AJ
Smith
A G
AG
Mountford
P S
PS
Burt
D W
DW
D001665:Binding Sites; D002277:Carcinoma; D004261:DNA Replication; D004352:Drug Resistance, Microbial; D004686:Encephalomyocarditis virus / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis* / Q000235:genetics; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics*; D008175:Lung Neoplasms; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D018741:Replication Origin / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D019309:Epstein-Barr Virus Nuclear Antigens; D005839:Gentamicins; D012333:RNA, Messenger; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); C046331:phosphoglucokinase; D019868:Kanamycin Kinase