Mutant/mutation
The mutant expresses the reporter protein β-Galactosidase under the control of the CSP promoter.

Protein (function)
The ability of E. coli β-galactosidase to hydrolyze β-D-galactopyranoside analogs, such as X-Gal, CPRG, or MUG, allows reliable quantification of reporter enzyme expression over a wide range using simple colorimetric assays.

Phenotype
Phenotype analyses indicate that expression of β-galactosidase does not affect the viability and infectivity of sporozoites. Oocysts and sporozoites express β-galactosidase. Expression of β-galactosidase can be visualised and quantified using simple enzymatic assays. Sporozoite loads can be quantified using the β-D-galactopyranoside analog CPRG. Liver stage loads could be quantified using two sensitive methods, the substrate MUG and a β-galactosidase ELISA.

Additional information
A monoclonal anti-β-Galactosidase antibody from Promega was used.

X-gal staining was performed on fixed sporozoites. Sporozoites were immobilised and permeabilized for 10 min at room temperature with 2% formaldehyde, 0.2% glutaraldehyde, 0.02% Triton-X 100 in phosphate buffered saline (PBS). After washing with PBS the parasites were incubated with staining solution (5 mM potassium ferricyanid, 5 mM potassium ferrcyanid, 0.04% deoxycholate, 2 mM magnesium chloride in phosphate buffered saline) containing 1 mg ml−1 X-Gal until appearance of staining.

Different quantitative β-Gal assays were used to determine levels of β-Galactosidase in sporozoites and liver stages.

Other mutants