Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Exp Parasitol. 2013 Jul;134(3):275-80. doi: 10.1016/j.exppara.2013.03.015. Epub 2013 Mar 29.
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.
在活体脑组织中检测刚地弓形虫从速殖子阶段到缓殖子阶段的转化非常困难，因为寄生虫体积小，且转化和再激活是短暂的事件。为了更好地理解刚地弓形虫速殖子和缓殖子之间的阶段转化机制，并在中间宿主中识别阶段转化，我们构建了刚地弓形虫的转cyst 形成株（PLK）。寄生虫在速殖子阶段稳定表达增强型绿色荧光蛋白（EGFP），在缓殖子阶段表达红色荧光蛋白（RFP），分别受 SAG1 和 BAG1 启动子的控制。由此产生的转基因寄生虫被命名为 PLK/Bi。PLK/Bi 速殖子在体外和体内仅在速殖子阶段表达绿色荧光，仅在缓殖子阶段表达红色荧光。荧光分析表明，重组 GFP 和 RFP 定位于细胞内的空泡空间。此外，我们还对转基因弓形虫的体外生长和培养条件进行了分析，并在体内评估了其毒力。我们的数据表明，PLK/Bi 的阶段特异性荧光表达可合理设计用于刚地弓形虫的体外和体内阶段转化和再激活研究。
