Molecular Genetic Tools for Parasitic Helminths

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。由于这些感染，5亿人遭受衰弱，有时是致命的疾病，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，迫切需要针对它们的新治疗和预防措施，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。缺乏现代分子工具来询问寄生蠕虫的基因功能和由此产生的程度，蠕虫生物学仍然是描述性的，而不是机械的性质，无疑是这种下降的因素。然而，自由生活的蠕虫秀丽隐杆线虫和真涡虫可以使用先进的分子工具进行研究，这表明开发类似的工具寄生虫是可行的。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，重大的技术障碍仍然存在，如果分子遗传学成为常规的蠕虫寄生虫学。因此，我们建议进一步开发和完善蠕虫寄生虫基因表达的操纵工具。我们将集中在两个重要的人类病原体，粪类圆线虫和曼氏血吸虫。它们分别代表线虫和扁形动物，这是医学上重要的两门蠕虫。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，允许在瞬时转化的S. mansoni和St. stercoralis。此外，我们将寻求确定调节序列，允许条件转基因表达和重组蛋白质的细胞内和细胞外运输。我们还将开发通过连续宿主传代建立稳定表达转基因株系的方法。将开发用于转基因的染色体整合的新方法，并且将鉴定绝缘子序列作为在瞬时和稳定转化的蠕虫中对抗表观遗传转基因沉默的措施。在我们的第二个具体目标下，我们将靶向表达基因以沉默S。mansoni和St. stercoralis。推测与C.线虫RDE-1、SAGO-1、SID-1和SID-2限制了外源施用dsRNA在St. stercoralis中的沉默效率，并且施用到宿主的siRNA可以沉默S.在此目的下，将在体内对曼氏血吸虫和St. stercoralis进行测试。拟议的研究有可能创造新的工具，将振兴被忽视的重要人类病原体的分子和细胞生物学研究。本申请提出开发新的方法来研究基因功能的分子水平上的血吸虫和粪类圆线虫，代表两个主要群体的寄生蠕虫（蠕虫）。其两个具体目标是发展S. mansoni和St. stercoralis的转基因，所述转基因能够调节组织特异性表达并且能够通过连续宿主传代稳定表达，以及用于靶向沉默或破坏两种寄生虫中的基因表达。由于这种性质的可靠技术要么不发达，要么完全不可用，拟议的项目有可能振兴寄生蠕虫学领域，这是目前被认为是主要描述性的，而不是机械的，在性质上。

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.