title: [Stable green fluorescent protein expression in Toxoplasma gondii mutant].
abstract: 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.
  
  
  CONCLUSIONS
  T. gondii mutant with GFP tag is constructed, which provides a new method to determine the proliferation when cultured in host cells.
authors.forename: Liang
authors.initials: L
authors.lastname: Wu
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Yuan
authors.forename: Xu-Gan
authors.initials: XG
authors.lastname: Jiang
authors.forename: Xing-Li
authors.initials: XL
authors.lastname: Fu
authors.forename: Zhao-Xi
authors.initials: ZX
authors.lastname: Lu
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Tu
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Sheng-Xia
authors.initials: SX
authors.lastname: Chen
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
mesh_terms: D005260:Female; D005434:Flow Cytometry; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D014122:Toxoplasma / Q000235:genetics* / Q000472:pathogenicity*; D014162:Transfection
chemical_list: D049452:Green Fluorescent Proteins