Department of Biochemistry and Molecular Biology, The Huck Center for Malaria Research, Pennsylvania State University, University Park, PA, USA.
Malar J. 2020 Nov 23;19(1):424. doi: 10.1186/s12936-020-03498-w.
Well-defined promoters are essential elements for genetic studies in all organisms, and enable controlled expression of endogenous genes, transgene expression, and gene editing. Despite this, there is a paucity of defined promoters for the rodent-infectious malaria parasites. This is especially true for Plasmodium yoelii, which is often used to study the mosquito and liver stages of malarial infection, as well as host immune responses to infection.
Here six promoters were selected from across the parasite's life cycle (clag-a, dynein heavy chain delta, lap4, trap, uis4, lisp2) that have been invoked in the literature as controlling their genes in a stage-specific manner. A minimal promoter length for the constitutive pybip promoter that confers strong expression levels was also determined, which is useful for expression of reporters and gene editing enzymes.
Instead, it was observed that these promoters confer stage-enriched gene control, as some parasites also effectively use these promoters in other stages. Thus, when used alone, these promoters could complicate the interpretation of results obtained from promoter swaps, stage-targeted recombination, or gene editing experiments.
Together these data indicate that achieving stage-specific effects, such as gene editing, is likely best done using a two-component system with independent promoter activities overlapping only in the intended life cycle stage.
明确的启动子是所有生物体中遗传研究的基本要素，可实现内源性基因的表达控制、转基因表达和基因编辑。尽管如此，对于啮齿动物感染性疟原虫，仍然缺乏明确的启动子。这在疟原虫 yoelii 中尤其如此，它通常用于研究疟原虫感染的蚊子和肝脏阶段，以及宿主对感染的免疫反应。
在这里，从寄生虫的整个生命周期中选择了六个启动子（clag-a、dynein heavy chain delta、lap4、trap、uis4、lisp2），这些启动子在文献中被认为以特定阶段的方式控制其基因。还确定了具有强表达水平的组成型 pybip 启动子的最小启动子长度，这对于报告基因和基因编辑酶的表达非常有用。
然而，观察到这些启动子赋予了阶段特异性的基因控制，因为一些寄生虫也在其他阶段有效地使用这些启动子。因此，当单独使用时，这些启动子可能会使从启动子替换、阶段靶向重组或基因编辑实验中获得的结果的解释复杂化。
总之，这些数据表明，要实现特定阶段的效果，例如基因编辑，最好使用具有独立启动子活性的两部分系统，这些活性仅在预期的生命周期阶段重叠。
