|Title||Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.|
|Publication Type||Journal Article|
|Year of Publication||1997|
|Authors||Ramage AD, Clark AJ, Smith AG, Mountford P, Burt DW|
|Date Published||1997 Sep 15|
|Keywords||Binding Sites, Carcinoma, DNA Replication, Drug Resistance, Microbial, Encephalomyocarditis virus, Epstein-Barr Virus Nuclear Antigens, Genes, Genetic Vectors, Gentamicins, Humans, Kanamycin Kinase, Lung Neoplasms, Phosphotransferases (Alcohol Group Acceptor), Promoter Regions, Genetic, Replication Origin, Ribosomes, RNA, Messenger, Transcription, Genetic, Transfection, Tumor Cells, Cultured|
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.