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Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors
Author(s) -
Tabotta Walter,
Klein Dieter,
Hohenadl Christine,
Salmons Brian,
Günzburg Walter H.
Publication year - 2001
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.209
Subject(s) - biology , transfection , green fluorescent protein , transcription (linguistics) , viral vector , microbiology and biotechnology , expression cassette , gene , cell culture , reporter gene , retrovirus , sumo protein , long terminal repeat , vector (molecular biology) , gene expression , recombinant dna , ubiquitin , genetics , linguistics , philosophy
Background A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. Methods In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution 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 enhanced 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 the 5′ LTR of the vector. Results PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence‐activated cell sorting (FACS). After detection of low‐level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3′ LTR at the R/U5 border to prevent accidental read‐through transcription from neighbouring cellular promoters. Virus‐containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells. Conclusions This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell. Copyright © 2001 John Wiley & Sons, Ltd.