Striepen B, He C Y, Matrajt M, Soldati D, Roos D S
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA. striepen@sas .upenn.edu
Mol Biochem Parasitol. 1998 May 1;92(2):325-38. doi: 10.1016/s0166-6851(98)00011-5.
We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).
我们构建了绿色荧光蛋白GFP的突变体（Cormack等人，在大肠杆菌中筛选出具有明亮荧光的突变体。基因，1996年；173:33 - 38），用于在原生动物寄生虫刚地弓形虫中表达。尽管完整的GFP未在任何可检测水平表达，但GFP融合蛋白可通过荧光显微镜、流式细胞术（FACS）和免疫印迹检测到。细胞外速殖子和刚地弓形虫感染的宿主细胞都可通过FACS轻松分选，这应有助于多种选择策略。测试了几种选择标记产生稳定绿色转基因寄生虫的能力。荧光强度与基因拷贝数和蛋白质表达水平直接相关。因此，由SAG1启动子驱动的弱选择标记如氯霉素乙酰转移酶（CAT），可产生多拷贝插入，对于选择绿色荧光寄生虫最为有效，特别是当与采用强启动子驱动GFP表达的构建体结合时。在这项工作过程中开发的转化载体对于在弓形虫中过表达异源转基因应具有普遍用途。CAT - GFP融合蛋白在寄生虫细胞质中表达。与P30主要表面抗原的GFP融合蛋白（在同一质粒上与受各种启动子控制的CAT选择标记相连）可在活细胞的致密颗粒中检测到，并有效分泌到寄生泡中。与棒状体蛋白ROP1的GFP融合蛋白靶向棒状体（寄生虫顶端的特殊分泌细胞器）。
