Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands.
Nuffield Department of Medicine, The Jenner Institute, University of Oxford, Oxford, United Kingdom.
Front Cell Infect Microbiol. 2019 Apr 16;9:96. doi: 10.3389/fcimb.2019.00096. eCollection 2019.
Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic (Pf) NF54 line was generated that expresses a fusion of and genes under the control of the .3 gene promoter (line mCherry-luc@etramp10.3). .3 is related to rodent and the promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive and promoters. The parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.
表达荧光和生物发光蛋白的转基因疟原虫是研究疟原虫生物学和评估药物和疫苗的有价值的工具。使用 CRISPR/Cas9 方法，生成了一种表达 和 基因融合的转基因 (Pf) NF54 系，该融合基因受.3 基因启动子（mCherry-luc@etramp10.3 系）的控制。.3 与啮齿动物 有关，并且 启动子已被用于驱动啮齿动物寄生虫子孢子和肝期的高转基因表达。我们在整个完整的生命周期中检查了转基因表达，并将其与表达 mCherry-荧光素酶和 GFP-荧光素酶的转基因系进行了比较，这些转基因系受组成型 和 启动子的控制。 寄生虫在配子体、子孢子和肝期表达 mCherry。虽然在无性血期寄生虫中未检测到高于背景水平的 mCherry 信号，但在无性血期、配子体、子孢子和肝期均检测到荧光素酶表达，其中 III-V 期配子体和子孢子中检测到的报告基因表达水平最高。mCherry 和荧光素酶在配子体和子孢子中的表达使这种转基因寄生虫系适合用于检查传播阻断抑制剂效果的 测定，并分析配子体和子孢子生物学。
