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.