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
Ta. Pfeifer et al., 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, Gene, 188(2), 1997, pp. 183-190
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.