Molecular Genetic Tools for Parasitic Helminths

寄生蠕虫的分子遗传学工具

• 批准号: 8260372
• 负责人: JAMES B LOK
• 金额: $ 38.59万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260372
• 关键词: Biological; Biology; Caenorhabditis elegans; Childhood; Cognitive; Communities; DNA; Developing Countries; Development; Double-Stranded RNA; Enzymes; Epigenetic Process; Funding; Gene Expression; Gene Silencing; Gene Transfer Techniques; Genes; Goals; Health; Helminths; Human; Infection; Intervention; Knowledge; Life; Measures; Medical; Methodology; Methods; Molecular; Molecular Genetics; Nature; Nematoda; Parasites; Parasitic nematode; Parasitology; Platyhelminths; Process; Public Health; RNA Interference; Recombinant Proteins; Research; Research Personnel; Resistance; Schistosoma mansoni; Science; Scientist; Strongyloides stercoralis; Techniques; Testing; Tissues; Transgenes; Transgenic Organisms; Vaccines; Veins; Work; extracellular; gene function; global health; in vivo; neglect; new technology; parasitism; pathogen; success; tool; transgene expression

DESCRIPTION (provided by applicant): Helminth (worm) parasites take an enormous toll on human health, especially in developing countries. Half a billion people suffer debilitating, sometimes fatal illness as a result of these infections, and subclinical effects of helminth parasitism include retarded physical and cognitive development. Despite their global health impact, and the pressing need for new treatments and preventative measures against them, there has been a decline in the numbers of investigators funded to work on these pathogens, and a consequent reduction in the number of young scientists choosing to enter this field. The paucity of modern molecular tools for interrogating gene function in parasitic helminths and the resulting degree to which helminth biology remains descriptive as opposed to mechanistic in nature are undoubtedly factors in this decline. Nevertheless, the free-living worm Caenorhabditis elegans and planaria can be studied using sophisticated molecular tools, suggesting that development of similar tools for parasitic worms is feasible. In this vein, we and others have had recent encouraging success in the experimental manipulation of gene expression in parasitic worms. Despite this, significant technical hurdles remain if molecular genetics is to become routine in helminth parasitology. Therefore, we propose to further develop and refine tools for the manipulation of gene expression in helminth parasites. We will focus on two important human pathogens, Strongyloides stercoralis and Schistosoma mansoni. These represent, respectively, the Nematoda and Platyhelminthes, two phyla of medically important helminths. The first of our two specific aims is to develop transgenic parasitic helminths that are amenable to experimentation. Work towards this aim will involve developing DNA constructs that allow regulated, tissue-specific transgene expression in transiently transformed S. mansoni and St. stercoralis. Also, we will seek to identify regulatory sequences that allow conditional transgene expression and both intra- and extracellular transport of recombinant proteins. We will also develop methods for establishing stably expressing transgenic lines through serial host passage. New methods for chromosomal integration of transgenes will be developed and insulator sequences will be identified as measures against epigenetic transgene silencing in both transiently and stably transformed worms. Under our second specific aim we will target expressed genes for silencing in S. mansoni and St. stercoralis. Hypotheses that the activities of RNAi processing enzymes homologous to C. elegans RDE-1, SAGO-1, SID-1 and SID-2 limit the silencing efficiency of exogenously applied dsRNA in St. stercoralis and that siRNAs administered to the host can silence genes of S. mansoni and St. stercoralis in vivo are to be tested under this aim. The proposed research has the potential to create new tools that will invigorate the molecular and cellular biological study of a neglected and important group of human pathogens. This application proposes to develop new methods to study gene function at the molecular level in Schistosoma mansoni and Strongyloides stercoralis, representatives of two major groups of parasitic helminths (worms). Its two specific aims propose to develop methods for DNA transformation of S. mansoni and St. stercoralis with transgenes capable of regulated tissue specific expression and of stable expression through serial host passage, and for targeted silencing or disruption of gene expression in the two parasites. Because reliable techniques of this nature are either underdeveloped or altogether unavailable, the proposed project has the potential to invigorate the field of parasitic helminthology, which is currently perceived as largely descriptive, as opposed to mechanistic, in nature.

描述（由申请人提供）：蠕虫（蠕虫）寄生虫对人类健康造成巨大损失，特别是在发展中国家。十亿人因这些感染而罹患使人衰弱、有时甚至致命的疾病，而蠕虫寄生的亚临床影响包括身体和认知发育迟缓。尽管它们对全球健康产生影响，并且迫切需要针对它们的新治疗方法和预防措施，但资助研究这些病原体的研究人员数量有所减少，选择进入这一领域的年轻科学家数量也随之减少。缺乏现代分子工具来探究寄生蠕虫的基因功能，以及由此导致的蠕虫生物学仍然是描述性的，而不是本质上的机械性，这无疑是导致这种下降的因素。尽管如此，可以使用复杂的分子工具来研究自由生活的线虫秀丽隐杆线虫和涡虫，这表明开发用于寄生虫的类似工具是可行的。本着这一精神，我们和其他人最近在寄生虫基因表达的实验操作方面取得了令人鼓舞的成功。尽管如此，如果分子遗传学要成为蠕虫寄生虫学的常规方法，仍然存在重大的技术障碍。因此，我们建议进一步开发和完善用于操纵蠕虫寄生虫基因表达的工具。我们将重点关注两种重要的人类病原体：粪类圆线虫和曼氏血吸虫。它们分别代表线虫纲和扁形动物纲，这两个医学上重要的蠕虫门。我们的两个具体目标中的第一个是开发适合实验的转基因寄生蠕虫。实现这一目标的工作将涉及开发 DNA 构建体，以允许在瞬时转化的曼氏链球菌和粪圆线虫中进行受调节的组织特异性转基因表达。此外，我们将寻求鉴定允许条件转基因表达以及重组蛋白的细胞内和细胞外运输的调控序列。我们还将开发通过连续宿主传代建立稳定表达转基因系的方法。将开发转基因染色体整合的新方法，并将鉴定绝缘子序列，作为对抗瞬时和稳定转化蠕虫中表观遗传转基因沉默的措施。根据我们的第二个具体目标，我们将针对曼氏链球菌和粪圆线虫中的表达基因进行沉默。假设与线虫 RDE-1、SAGO-1、SID-1 和 SID-2 同源的 RNAi 加工酶的活性限制了圆线虫中外源应用的 dsRNA 的沉默效率，并且给予宿主的 siRNA 可以在体内沉默曼氏圆线虫和圆线虫的基因，这些假设将在此目的下进行测试。拟议的研究有可能创造新的工具，从而激发对被忽视的重要人类病原体的分子和细胞生物学研究。本申请提出开发新方法，在分子水平上研究曼氏血吸虫和粪类圆线虫（两大寄生蠕虫（蠕虫）的代表）的基因功能。其两个具体目标是开发曼氏链球菌和粪圆线虫的 DNA 转化方法，其转基因能够调节组织特异性表达并通过连续宿主传代稳定表达，并有针对性地沉默或破坏这两种寄生虫的基因表达。由于这种性质的可靠技术要么不发达，要么完全不可用，因此拟议的项目有可能振兴寄生蠕虫学领域，目前寄生蠕虫学本质上被认为主要是描述性的，而不是机械性的。