GENERATION OF A CONDITIONALLY NEO(R)-CONTAINING RETROVIRAL PRODUCER CELL-LINE - EFFECTS OF NEO(R) ON RETROVIRAL TITER AND TRANSGENE EXPRESSION

We have developed a method for generating high-titer retroviral produc er cell lines conditionally containing a neomycin resistance gene (neo (r)) based on the Cre/loxP system. For this, a bicistronic retroviral splicing vector carrying the green fluorescence protein (GFP) and a ma rker gene cassette consisting of internal ribosome entry site (IRES) a nd neo flanked by loxP sites, was constructed and conveniently used to generate a G418 resistant vector produced cell line. Following titer determination and verification of the biological activity of the retro viral supernatants, the selectable expression cassette which was no lo nger required was excised from the provirus by transient Cre expressio n using an adenoviral vector. This strategy led to precise excision of neo(r) and generation of retroviral supernatants containing functiona l 'neo-less' retroviral particles without detrimental effects on the h igh vector titers found in the parental neo(r)-containing producer lin es. GFP expression was significantly increased after the excision of n eo, in both the producer lines and retrovirally transduced target cell s. Reintroduction of neo did not alter GFP expression, suggesting that the neo gene and/or its gene product per se are not acting as a trans criptional silencer.