Vinayak Sumiti, Brooks Carrie F, Naumov Anatoli, Suvorova Elena S, White Michael W, Striepen Boris
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
Departments of Molecular Medicine & Global Health, University of South Florida, Tampa, Florida, USA.
mBio. 2014 Dec 2;5(6):e02021. doi: 10.1128/mBio.02021-14.
Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.
Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.
顶复门原虫是专性细胞内寄生虫，可在人类和动物中引发重要疾病。操纵病原体基因组是了解特定基因在寄生虫发育和发病机制中功能的最直接方法。在刚地弓形虫中，非同源重组通常比同源重组更受青睐，而同源重组是精确基因靶向所需的过程。已经开发了几种方法来克服这个问题，包括使用具有大片段侧翼以驱动位点特异性重组的靶向载体。我们构建了一个新的刚地弓形虫基因组DNA大插入文库，该文库经过排列和测序，覆盖了该寄生虫所有基因的95%。来自这个fosmid文库的克隆以单拷贝形式保存，这提供了高度的稳定性，并极大地增强了我们对该生物体进行改造的能力。我们建立了一个强大的重组工程流程，并表明我们的fosmid克隆可以在一个4天的方案中轻松转化为基因敲除构建体，该方案不需要基于平板的克隆，而是可以在多孔板中进行。我们验证了这种方法以了解刚地弓形虫中的基因功能，并产生了一个属于NOL1/NOP2/SUN家族的核仁蛋白的条件性无效突变体，并且我们表明该基因对寄生虫生长至关重要。我们还在化学诱变和全基因组测序的背景下展示了一种强大的互补策略。这个文库是一个重要的新资源，将加速对这种重要病原体的正向和反向遗传分析。
刚地弓形虫是理解细胞内寄生现象的重要遗传模型。我们在此表明，大插入基因组克隆是增强同源重组并使我们能够构建条件性突变体以了解基因功能的有效工具。我们构建了一个在拷贝控制载体中的刚地弓形虫基因组DNA的fosmid文库，该文库经过排列和测序，对基因组具有出色的覆盖度。fosmids以单拷贝状态保存，这极大地提高了它们的稳定性，并允许通过一个简单且高度可扩展的方案进行改造。我们在此表明，经过改造和未改造的fosmid克隆是正向和反向遗传学的强大工具。
