[Stable green fluorescent protein expression in Toxoplasma gondii mutant]
Objective:
To construct a Toxoplasma gondii mutant for stably expressing green fluorescent protein (GFP), and establish method to determine the rate of mutant-infected HeLa cells.
Methods:
Freshly lysed-out tachyzoites of T. gondii RH strain were transfected with plasmid ptubP30-GFP/sag-CAT. Stable transformants were selected with chloramphenicol and limited dilution. The expression of GFP in mutant tachyzoite was determined by RT-PCR and fluorescence microscopy. When infected with 1 x 10(4)-1 x 10(7) mutant tachyzoites respectively for 24 h, the total number of HeLa cells with green fluorescence was determined by fluorescent microscope in 10 high-power fields, and the rate of HeLa cells with parasitophorous vacuole was determined by flow cytometry.
Results:
Untransfected tachy-zoites were killed by chloramphenicol, while the stable transformants showed resistance to chloramphenicol. The expression of GFP gene was detected by RT-PCR. The P30-GFP transfectants displayed fluorescence outside the parasite. The rate of mutant-infected HeLa cells increased with the rise of the number of mutant for infection. When infected with 1 x 10(4)-1 x 10(7) tachyzoites, the numbers of HeLa cells with fluorescence were (14 +/- 6), (133 +/- 45), (332 +/- 93) and (443 +/- 90), and the rates of infected cells were (0.49 +/- 0.09)%, (8.76 +/- 0.50)%, (21.0 +/- 21.49)%, and (39.00 +/- 3.47)% by flow cytometry, respectively.
Conclusion:
T. gondii mutant with GFP tag is constructed, which provides a new method to determine the proliferation when cultured in host cells.
