title: Determination of stage interconversion in vitro and in vivo by construction of transgenic Toxoplasma gondii that stably express stage-specific fluorescent proteins.
abstract: Detection of Toxoplasma gondii conversion from the tachyzoite stage to the bradyzoite stage in living brain tissue is difficult because the parasites are small and conversion and reactivation of the parasites are transient events. To better understand the mechanisms of T. gondii stage conversion between tachyzoites and bradyzoites, and to recognize stage conversion in an intermediate host, we constructed a transgenic cyst-forming strain (PLK) of T. gondii. The parasites stably expressed enhanced green fluorescence protein (EGFP) in the tachyzoite stage and red fluorescence protein (RFP) in the bradyzoite stage, under the control of the SAG1 and BAG1 promoters, respectively. The resulting transgenic parasite was designated as PLK/Bi. The PLK/Bi zoites expressed only green fluorescence in the tachyzoite stage and only red fluorescence in the bradyzoite stage in vitro and in vivo. Fluorescence analyses showed that recombinant GFP and RFP were located to the intracellular vacuolar spaces. In addition, an analysis of growth and culture conditions of transgenic T. gondii was performed in vitro and the virulence was evaluated in vivo. Our data suggested that the stage-specific fluorescence expression by PLK/Bi may be rationally designed for in vitro and in vivo studies on stage conversion and reactivation of T. gondii.
authors.affiliation: Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Houshuang
authors.initials: H
authors.lastname: Zhang
authors.forename: Yanlei
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Cao
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Na
authors.initials: N
authors.lastname: Wang
authors.forename: Jinlin
authors.initials: J
authors.lastname: Zhou
mesh_terms: D000818:Animals; D001921:Brain / Q000469:parasitology; D002522:Chlorocebus aethiops; D018274:Electroporation; D005260:Female; D005347:Fibroblasts / Q000469:parasitology; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D030781:Organisms, Genetically Modified; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism / Q000472:pathogenicity; D014124:Toxoplasmosis, Animal / Q000469:parasitology; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism; D014709:Vero Cells; D014774:Virulence; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D000097573:Red Fluorescent Protein