Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.
Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.
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DOI：
10.1371/journal.ppat.1009224
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发表时间：
2021-01
期刊：
影响因子：
6.7
通讯作者：
Wickstead B
中科院分区：
文献类型：
--
作者：
Awuah-Mensah G;McDonald J;Steketee PC;Autheman D;Whipple S;D'Archivio S;Brandt C;Clare S;Harcourt K;Wright GJ;Morrison LJ;Gadelha C;Wickstead B
非洲动物锥虫病 (AAT) 是一种严重的家畜和多种野生动物的消耗性疾病。牛的疾病每年导致数百万头动物死亡，给撒哈拉以南非洲的农业造成重大经济损失。牛 AAT 主要由原生动物寄生虫刚果锥虫和间日锥虫引起，但对这些生物体致病阶段的实验室研究因难以进行哪怕是微小的基因改造而受到严重抑制。因此，有关这些寄生虫生物学的许多重要的基本问题无法得到解决。在这里，我们证明刚果锥体基因组参考菌株的体外培养物可以直接以血流形式可靠且高效地进行修饰。我们描述了表达刚果锥虫优化的 T7 RNA 聚合酶和 Tet 阻遏蛋白的亲本单标记系，并表明微型染色体位点可用作在非诱导细胞中具有低背景的稳定、可调节转基因表达的位点。使用这些工具，我们描述了用于诱导性 RNA 干扰 (RNAi) 的生物体特异性构建体，并证明了多个必需和非必需基因的敲低。我们还表明，可以利用微型染色体位点来创建稳定的血流形式细胞系，每次转染可以稳健地提供超过 40,000 个独立的稳定克隆，从而能够产生基因组规模的高复杂性文库。最后，我们证明刚果锥虫的修饰形式仍然具有传染性，创建了可用于 AAT 模型的稳定的高生物发光线，并通过体内成像跟踪小鼠的感染过程。这些实验建立了一套基本工具，将刚果锥虫从技术上具有挑战性的生物体转变为功能遗传学的常规模型，并使我们能够开始解决有关这种重要寄生虫生物学的一些基本问题。寄生虫刚果锥虫和间日锥虫是非洲动物锥虫病 (AAT) 最重要的病原体。 AAT 每年大约杀死 300 万头牛，给撒哈拉以南非洲地区的粮食生产带来巨大的经济负担。了解病原体生物学的一个关键工具是进行基因修饰的能力，特别是创建目标基因的特定突变体，可用于研究基因产物的位置、表达变化的影响或完全基因去除的后果。然而，AAT 的工作受到了严重限制，因为即使是很小的基因修饰也很困难，并且缺乏许多功能遗传学应用的工具。在这里，我们为刚果锥虫设计、测试和验证了一套工具，这些工具首次带来了：常规高效基因标记和敲除、沉默位点的可调节转基因表达、用于诱导基因敲除的物种特异性系统、用于体内疾病模型的生物发光系，以及生成高度复杂的突变体文库的方法，从而实现基因组规模的工作。这些数据和相关工具将极大地帮助 AAT 和刚果锥虫生物学的研究。
Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection–enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite. The parasites Trypanosoma congolense and T. vivax are the most significant causative agents of Animal African trypanosomiasis (AAT). AAT kills an estimated 3 million cattle each year and represents a huge financial burden on food production in sub-Saharan Africa. A critical tool for understanding pathogen biology is the ability to make genetic modifications, especially creating specific mutants of target genes that can be used to investigate the locations of gene products, the effects of changes in expression, or consequence of complete gene removal. However, work on AAT is severely limited by difficulties in making even small genetic modifications and lack of tools for many functional genetics applications. Here, we design, test and validate a set of tools for T. congolense that brings for the first time: routine high-efficiency gene tagging and knockout, regulatable transgene expression from silent loci, a species-specific system for inducible gene knockdown, bioluminescent lines for in vivo disease models, and a means to generate highly complex libraries of mutants that will enable genome-scale work. These data and the tools around them will greatly aid research into AAT and T. congolense biology.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
2.4
作者：
HEMPHILL, A;FRAME, I;ROSS, CA
通讯作者：
ROSS, CA
DOI：
10.1111/j.1550-7408.1978.tb04405.x
发表时间：
1978-01-01
期刊：
JOURNAL OF PROTOZOOLOGY
影响因子：
--
作者：
BANKS, KL
通讯作者：
BANKS, KL
影响因子：
1.5
作者：
Biebinger, S;Wirtz, LE;Clayton, C
通讯作者：
Clayton, C
影响因子：
3.3
作者：
Abbas AH;Silva Pereira S;D'Archivio S;Wickstead B;Morrison LJ;Hall N;Hertz-Fowler C;Darby AC;Jackson AP
通讯作者：
Jackson AP