B. Striepen et al., EXPRESSION, SELECTION, AND ORGANELLAR TARGETING OF THE GREEN FLUORESCENT PROTEIN IN TOXOPLASMA-GONDII, Molecular and biochemical parasitology, 92(2), 1998, pp. 325-338
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
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