Developing tools for genetic manipulation of hookworms

开发钩虫基因操作工具

• 批准号: 8649018
• 负责人: JOHN M HAWDON
• 金额: $ 18.35万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649018
• 关键词: Adult; Anemia; Antibiotic Resistance; Antibiotics; Antigen Targeting; Area; Biological Assay; Biology; Brugia malayi; Caenorhabditis elegans; Cell Culture Techniques; Chemicals; Child; Chromosomes; Communicable Diseases; Data; Development; Disease; Drug Targeting; Drug resistance; Elderly; Embryo; Environment; Exploratory/Developmental Grant; Female of child bearing age; Foundations; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Transfer Techniques; Genes; Genetic; Genetic Vectors; Genome; Genomics; Gold; Growth; Helminths; Hemorrhage; Hookworm Infections; Hookworms; In Vitro; Individual; Infection; Intestines; Investigation; Iron deficiency anemia; Larva; Lead; Life; Life Cycle Stages; Lipids; Luciferases; Mammalian Cell; Mediating; Methods; Modeling; Molecular Genetics; Molting; Morbidity - disease rate; Nature; Necator americanus; Nematoda; Old World Hookworm; Parasites; Parasitic infection; Parasitic nematode; Pharmaceutical Preparations; Population; Pregnant Women; Process; Public Health; Recombinant Vaccines; Reporter; Reporter Genes; Research; Retrotransposon; School-Age Population; Staging; System; Techniques; Technology; Time; Tissues; Transfection; Transgenes; Vaccine Antigen; Vaccines; World Bank; base; disability-adjusted life years; feeding; gene function; genetic manipulation; improved; novel; particle; promoter; public health relevance; tool; uptake; vector

DESCRIPTION (provided by applicant): Hookworm infection remains one of the greatest public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. Vaccine efforts suffer for the lack of effective target antigens. Development of new drug targets and improved vaccine antigens will require a better understanding of hookworm biology, particularly the infective process. However, the obligate requirement for a host and the inability to grow the complete life cycle in vitro precluded development of powerful genetic tools for hookworm research until recently. We propose to adapt and build upon recent advances using other parasitic nematodes to develop a transfection system for hookworms. We will take two parallel but independent approaches to develop transient transfection methods for hookworms that will provide a foundation for the future development of heritable transformation technology. In Aim 1, particle bombardment will be used to introduce reporter genes into embryos and larval stages, and transformants will be assayed for survival, growth and reporter gene expression. We will also use a recently described antibiotic selection technique to select transformants from non-transformants. In Aim 2, we will use chemical and lipid-based technology to introduce reporter genes into molting larvae, a technique that was recently successful in the parasitic nematode Brugia malayi. In both aims we will use a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into hookworm chromosomes. Development of in vitro transfection would represent a significant advance for hookworm research, and will allow determination of hookworm gene expression and function in a homologous genetic context for the first time. Furthermore, transgenesis would enable us to exploit the data from the imminent release of several sequenced hookworm genomes and transcriptomes for the development of novel hookworm control strategies.

描述（由申请人提供）：钩虫感染仍然是全世界最大的公共卫生威胁之一，估计有8亿人感染。严重的钩虫感染是热带地区贫血的主要原因，导致身体虚弱，有时甚至致命的缺铁性贫血，这是由于在肠道内喂养成虫时失血造成的。儿童、孕妇和老年人特别容易感染钩虫。控制策略仅限于对受感染个体进行定期除虫，这受到再感染率高和耐药蠕虫种群发展的限制。由于缺乏有效的靶抗原，疫苗工作受到影响。开发新的药物靶点和改进疫苗抗原将需要更好地了解钩虫生物学，特别是感染过程。然而，直到最近，对寄主的强制性要求和无法在体外生长完整的生命周期阻碍了用于钩虫研究的强大遗传工具的开发。我们建议适应和建立在最近的进展，利用其他寄生线虫来开发一个钩虫转染系统。我们将采用两种平行但独立的方法来开发钩虫瞬时转染方法，为未来遗传转化技术的发展奠定基础。在Aim 1中，将使用粒子轰击将报告基因引入胚胎和幼虫阶段，并检测转化子的存活、生长和报告基因表达。我们还将使用最近描述的抗生素选择技术从非转化体中选择转化体。在Aim 2中，我们将使用化学和基于脂质的技术将报告基因引入蜕皮幼虫，这项技术最近在寄生线虫布鲁吉亚马来身上取得了成功。在这两个目的中，我们将使用一个基于逆转录转座子的整合载体将报告基因引入钩虫染色体。体外转染技术的发展将是钩虫研究的重大进展，并将首次在同源遗传背景下确定钩虫基因的表达和功能。此外，转基因将使我们能够利用即将发布的几个钩虫基因组测序和转录组的数据来开发新的钩虫控制策略。

项目成果

Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: A new tool for anthelmintic research.

用于宿主阶段钩虫和猪蛔虫幼虫电生理记录的微流体平台：驱虫研究的新工具。

• DOI: 10.1016/j.ijpddr.2016.08.001
• 发表时间: 2016
• 期刊: International journal for parasitology. Drugs and drug resistance
• 作者: Weeks,JanisC;Roberts,WilliamM;Robinson,KristinJ;Keaney,Melissa;Vermeire,JonJ;UrbanJr,JosephF;Lockery,ShawnR;Hawdon,JohnM
• 通讯作者: Hawdon,JohnM