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 (beta Gal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker f or stable transformation. Three Tg expression vectors with different p romoters driving lacZ were constructed and shown in transient transfec tions to differ in their relative expression levels. Using a fluoresce nt beta Gal substrate, it was possible to detect enzymatic activity wi th as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microti ter plates in the presence of the color substrate, chlorophenol red-be ta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected para site cultures in the presence of CPRG, we were able to clone stably ex pressing beta Gal-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 beta Gal to studies on Tg provides not only a much needed second reporter for tran sient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.