BACULOVIRUS IMMEDIATE-EARLY PROMOTER-MEDIATED EXPRESSION OF THE ZEOCIN(TM) RESISTANCE GENE FOR USE AS A DOMINANT SELECTABLE MARKER IN DIPTERAN AND LEPIDOPTERAN INSECT-CELL LINES

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for u se as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kcl and SL2, was inhibited at Zeocin concentrations of 50 and 75 mu g/ml, respectively, while the S podoptera cell line, Sf9, was inhibited at a concentration of 250 mu g /ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (S V40) early promoters did not function in these insect cell lines. Seve ral baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autograph a californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to d rive expression of the Zeocin resistance gene (ble) in these cell line s. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presenc e of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promot ers, as demonstrated by P-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector , containing ten unique restriction sites, was constructed which allow s for selection of Zeocin resistance in insect cell lines and in Esche richia coli.