Molecular Genetic Tools for Parasitic Helminths

说明（由申请人提供）：蠕虫（蠕虫）寄生虫对人类健康造成巨大危害，特别是在发展中国家。由于这些感染，有5亿人患有身体虚弱，有时甚至是致命的疾病，寄生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有影响，而且迫切需要新的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，因此选择进入这一领域的年轻科学家数量也有所减少。用于研究寄生蠕虫基因功能的现代分子工具的缺乏，以及由此导致的蠕虫生物学在本质上仍然是描述性的，而不是机械性的，这些无疑是导致这种衰退的因素。然而，可以使用复杂的分子工具研究自由生活的秀丽隐杆线虫和涡虫，这表明开发类似的寄生虫工具是可行的。在这方面，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蠕虫寄生虫基因表达操作工具。我们将重点介绍两种重要的人类病原体，粪类圆线虫和曼氏血吸虫。这些分别代表了线虫和扁形蠕虫，两个医学上重要的蠕虫门。我们的两个具体目标的第一个是开发转基因寄生蠕虫，适用于实验。实现这一目标的工作将包括开发DNA结构，允许在瞬时转化的mansoni和St. stercoralis中进行调节，组织特异性转基因表达。此外，我们将寻求确定允许有条件的转基因表达和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寄主传代建立稳定表达转基因株系的方法。转基因染色体整合的新方法将被开发，绝缘子序列将被确定为防止瞬变和稳定转化蠕虫表观遗传转基因沉默的措施。在我们的第二个特定目标下，我们将针对S. mansoni和St. stercoralis的沉默表达基因。关于线虫RDE-1、SAGO-1、SID-1和SID-2同源的RNAi加工酶的活性限制了外源应用于St. stercoralis的dsRNA的沉默效率，以及给宿主sirna可以在体内沉默S. mansoni和St. stercoralis的基因的假设，将在此目的下进行验证。拟议的研究有可能创造新的工具，将激发一个被忽视的和重要的人类病原体组的分子和细胞生物学研究。本申请旨在开发新的方法在分子水平上研究曼氏血吸虫（Schistosoma mansoni）和粪圆形线虫（Strongyloides stercoralis）这两种主要寄生蠕虫类群的基因功能。该研究的两个具体目标是，利用能够通过宿主序列传代调节组织特异性表达和稳定表达的转基因基因对mansoni和St. stercoralis进行DNA转化，以及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，要么完全无法获得，因此拟议的项目有可能为寄生蠕虫学领域注入活力，该领域目前被认为在本质上主要是描述性的，而不是机械性的。

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.