Abstract：
Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.
摘要：
条件基因表达是研究假定的疫苗和药物靶标的有力工具，尤其是在诸如恶性疟原虫的单倍体生物中。基于任一转录调节的可诱导系统，翻译,蛋白质或mRNA稳定性，其中,允许打开或关闭任何所需基因的表达，从而导致功能表型的特定获得或丧失。然而,这些系统可能很麻烦，涉及构建大型质粒和产生多个转基因寄生虫系。此外,所实现的动态调节范围对于每个单独的基因是不可预测的,并且当目的基因沉默时可能不足以产生可检测的表型。这里,我们结合了三个不同的诱导系统来调节单个基因的表达。报道分子NanoLuc荧光素酶的表达受到40倍以上的调控，这与每个单独系统相互相乘所实现的调节相关。我们应用有条件表达的NanoLuc来评估速效抗疟药如氯喹和青蒿琥酯以及速效抗疟药如atovaquone的效果。有条件表达的报告分子可以更快，更可靠地检测对寄生虫的毒性，这与每种化合物的预期作用相关。因此，通过表达这种可诱导的高度敏感的报道分子而实现的生物发光是研究潜在新抗疟药物的时间效应的有希望的工具。这种单质粒组合系统也可能证明可用于实现目的基因的充分调节以产生功能丧失表型。
