PDF(Pubmed)
Abstract：
The skin-penetrating gastrointestinal parasitic nematode Strongyloides stercoralis causes strongyloidiasis, which is a neglected tropical disease that is associated with severe chronic illness and fatalities. Unlike other human-infective nematodes, S. stercoralis cycles through a single free-living generation and thus serves as a genetically tractable model organism for understanding the mechanisms that enable parasitism. Techniques such as CRISPR/Cas9-mediated mutagenesis and transgenesis are now routinely performed in S. stercoralis by introducing exogenous DNA into free-living adults and then screening their F1 progeny for transgenic or mutant larvae. However, transgenesis in S. stercoralis has been severely hindered by the inability to establish stable transgenic lines that can be propagated for multiple generations through a host; to date, studies of transgenic S. stercoralis have been limited to heterogeneous populations of transgenic F1 larvae. Here, we develop an efficient pipeline for the generation of stable transgenic lines in S. stercoralis. We also show that this approach can be used to efficiently generate stable transgenic lines in the rat-infective nematode Strongyloides ratti. The ability to generate stable transgenic lines circumvents the limitations of working with heterogeneous F1 populations, such as variable transgene expression and the inability to generate transgenics of all life stages. Our transgenesis approach will enable novel lines of inquiry into parasite biology, such as transgene-based comparisons between free-living and parasitic generations.
摘要：
皮肤穿透性的胃肠道寄生虫线虫会导致线虫病，这是一种被忽视的热带病，与严重的慢性疾病和死亡有关。不像其他感染人类的线虫,S.stercoralis通过单个自由生活的世代循环，因此可以作为遗传上易于处理的模型生物，用于理解导致寄生的机制。现在，通过将外源DNA引入自由生活的成虫中，然后筛选其F1后代中的转基因或突变幼虫，可以在Stercoralis中常规进行CRISPR/Cas9介导的诱变和转基因。然而,由于无法建立可以通过宿主繁殖多代的稳定转基因系，因此严重阻碍了S.stercoralis的转基因；迄今为止，转基因S.stercoralis的研究仅限于转基因F1幼虫的异质种群。这里,我们开发了一种有效的管道，用于在胸骨链球菌中产生稳定的转基因品系。我们还表明，这种方法可用于在大鼠感染的线虫Strongyloidesratti中有效产生稳定的转基因系。产生稳定的转基因品系的能力避免了与异质F1种群合作的局限性，例如可变的转基因表达和无法产生所有生命阶段的转基因。我们的转基因方法将使人们能够对寄生虫生物学进行新的研究，例如基于转基因的自由生活和寄生世代之间的比较。
