Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors

Background A major prerequisite for the design of retroviral vectors encodi ng cell toxic or harmful genes is the possibility to tightly control gene e xpression, thus limiting activity to the relevant target cells and protecti ng the packaging cell used for production of recombinant viral particles. Methods In the present study a system was developed in which genetic reshuf fling during the retroviral life cycle is exploited, allowing reconstitutio n of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus ( MLV)-based reconstituting viral vectors (ReCon), a promoterless inverted en hanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3' LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of t he 5' LTR of the vector. Results PA317 packaging cells stably transfected with ReCon vectors were es tablished and EGFP expression was analysed by fluorescence-activated cell s orting (FACS). After detection of low-level background expression, an addit ional polyadenylation signal was introduced in antisense orientation into t he 3' LTR at the R/U5 border to prevent accidental read-through transcripti on from neighbouring cellular promoters. Virus-containing cell culture supe rnatants were then used to infect NIH3T3 target cells. EGFP expression, rec loning and sequencing of integrated proviruses demonstrated the correct rea ssembly of the transduced ubiquitin/EGFP transcription unit in these infect ed cells. Conclusions This facile and convenient system should allow production of re troviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector product ion if expressed in the retroviral packaging cell. Copyright (C) 2001 John Wiley & Sons, Ltd.