Developing genetic tools for a new model of infection and immunity

描述（由申请人提供）：寄生线虫（PN）感染仍然是全球人类健康的主要威胁，感染人数超过10亿。儿童、孕妇和老年人特别容易因线虫感染而发病。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。目前还没有针对人类泌尿系膜感染的疫苗。新药和疫苗的开发将需要更好地了解PN生物学，特别是感染过程和宿主对感染的免疫反应。专性宿主的要求和缺乏良好的动物模型限制了对PNs感染和篡夺宿主免疫反应机制的研究。虽然自由生活的秀丽隐杆线虫是线虫发育的极好模型，但它不是寄生虫。昆虫寄生线虫Heterorhabditis bacteriophora （Hb）具有秀丽隐杆线虫的许多优点，包括易于培养和操作所有生活史阶段，作为一种易处理的PN感染模型提供了潜力。然而，许多强大的遗传工具，如转基因和RNA干扰，尚未在Hb中充分发展，限制了其作为广泛模型的使用。我们建议在Hb中开发这些遗传工具，重点针对感染性幼年期和早期寄生期，以研究感染机制。我们将采取两种平行但独立的方法来开发这些工具，这将为未来研究PN感染的机制奠定基础。在目标1中，我们建议通过种系显微注射一种基于逆转录转座子的整合载体来开发Hb的转染方案，将报告基因引入Hb染色体。在目标2中，我们将为第二代Hb雌雄同体开发一个强大的RNAi方案。通过关注第二代雌雄同体，我们理想地定位于探索PN感染的生物学，因为这一阶段的后代是感染阶段。在这种新型模型线虫中开发基因敲除（反向遗传学）和转染方法将代表PN研究的重大进展，并将首次研究PN基因在感染过程中的功能。

DESCRIPTION (provided by applicant): Parasitic nematode (PN) infections remain a major threat to human health worldwide, with more than 1 billion people infected. Children, pregnant women, and the elderly are particularly susceptible to morbidity from nematode 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. There are no vaccines available for PN infections in humans. Development of new drugs and vaccines will require a better understanding of PN biology, particularly the infective process and the host's immune response to infection. The requirement of an obligate host and the lack of good animal models have limited investigations into mechanisms by which PNs infect and usurp the host immune response. While the free living nematode Caenorhabditis elegans is an excellent model for nematode development, it is not a parasite. The insect parasitic nematode Heterorhabditis bacteriophora (Hb) offers potential as a tractable model of PN infection that has many of the advantages of C. elegans, including easy culture and manipulation of all life history stages. However, many of the powerful genetic tools, like transgenesis and RNA interference, have not been fully developed in Hb, restricting its use as a widespread model. We propose to develop these genetic tools in Hb with a focus on targeting the infective juvenile and early parasitic stages in order to investigate infection mechanisms. We will take two parallel but independent approaches to develop these tools that will provide a foundation for future investigation into the mechanism of PN infection. In Aim 1, we propose to develop a transfection protocol for Hb via germ-line microinjection of a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into Hb chromosomes. In Aim 2, we will develop a robust RNAi protocol for 2nd generation Hb hermaphrodites. By focusing on 2nd generation hermaphrodites we are ideally positioned to probe the biology of PN infection, as the progeny of this stage are the infective stage. Development of methods for gene knock down (reverse genetics) and transfection in this novel model nematode would represent a significant advance for PN research, and will allow investigation of PN gene function during infection for the first time.