IMPROVED EBV-BASED SHUTTLE VECTOR SYSTEM - DICISTRONIC MESSENGER-RNA COUPLES THE SYNTHESIS OF THE EPSTEIN-BARR NUCLEAR ANTIGEN-1 PROTEIN TONEOMYCIN RESISTANCE

Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an ori P-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 ge ne product can be toxic in some cell types and may be selected against . In this paper, we describe a gene construct that overcomes this limi tation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resis tance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely i ntegrated into human cell lines. The presence of EBNA-1 protein was re flected by a large increase in transfection frequencies (1000-fold) us ing 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 i ntegration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector s ystems and should result in its increased use. (C) 1997 Elsevier Scien ce B.V.