EXPRESSION, SELECTION, AND ORGANELLAR TARGETING OF THE GREEN FLUORESCENT PROTEIN IN TOXOPLASMA-GONDII

We have engineered a mutant version of the green fluorescent protein G FP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1 996;173:33-38) for expression in the protozoan parasite Toxoplasma gon dii. Although intact GFP was not expressed at any detectable level, GF P fusion proteins could be detected by fluorescence microscopy, how cy tometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectabl e markers were tested for their ability to produce stable green transg enic parasites. Fluorescence intensity was directly correlated with ge ne copy number and protein expression level. Weak selectable markers s uch as chloramphenicol acetyl transferase (CAT) driven by the SAG1 pro moter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Tr ansformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cyt oplasm. GFP fusions to the P30 major surface antigen (linked on the sa me plasmid to a CAT selectable marker under control of various promote rs) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secre tory organelles at the apical end of the parasite). (C) 1998 Elsevier Science B.V. All rights reserved.