Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii.
We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.
Seeber
F
F
Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305, USA.
Boothroyd
J C
JC
D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D006651:Histocytochemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014122:Toxoplasma / Q000235:genetics*; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics*
D015415:Biomarkers; D001616:beta-Galactosidase