STABLE DNA TRANSFECTION OF THE PRIMITIVE PROTOZOAN PATHOGEN GIARDIA-LAMBLIA

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran ge ne with a bacterial neomycin phosphotransferase gene (neo). This plasm id was electroporated into G. lamblia, and the transfectants were sele cted by G418. pRANneo replicated episomally to similar to 80 copies pe r G. lamblia trophozoite as demonstrated by dot hybridizations, Southe rn hybridizations and transformations of the DpnI-treated plasmids int o Escherichia coli. pRANneolGDHluc was then constructed by incorporati on of a luciferase expression system into pRANneo to persistently expr ess firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. Imablia cells by Western blottings. The level of luciferase activity and the plasmi d copy number correlated with the concentration of G418. Removal of G4 18 from the transfectant culture resulted in gradual loss of the plasm id and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia. (C) 1998 El sevier Science B.V. All rights reserved.