Transduction of Schistosoma mansoni by pseudotyped retrovirus

描述（由申请方提供）：培养的寄生虫体的逆转录病毒转导提供了一种建立寄生虫体转基因系的潜在方法，从而有助于阐明寄生虫基因功能和表达。这是我们研究的长期目标。我们建议修改莫洛尼鼠白血病逆转录病毒（MMLV）载体pLNHX纳入荧光素酶和其他报告基因的控制下的内源性染色体基因启动子。pLNHX构建体和编码水泡性口炎病毒糖蛋白（VSVG）的质粒将用于转染GP 2 -293细胞以产生用VSVG假型化的复制缺陷型逆转录病毒颗粒。目的1研究MMLV-VSVG逆转录病毒对曼氏血吸虫的杀伤能力。包括孢子囊、胚泡和成虫在内的胚泡体的发育阶段将暴露于逆转录病毒。通过与聚凝胺、磷脂酰丝氨酸孵育和/或通过离心，将促进逆转录病毒转导的核糖体。将通过VSVG和逆转录病毒衣壳蛋白的免疫荧光共定位和超微结构技术来研究病毒结合和摄取到被膜的早期阶段。将通过Southern杂交分析、反向PCR和相关程序、免疫印迹和报告蛋白测定研究下游事件，包括将前病毒形式的逆转录病毒转基因整合到染色体中、从整合的报告基因转录以及翻译的报告蛋白的活性。在目标2中，我们将用编码基因特异性双链RNA的转基因盒修饰的MMLV-VSVG病毒体（而不是如目标1中的报告基因，如荧光素酶）转染病毒体。我们将研究这种转导是否是可遗传的，以及它是否会导致模型靶基因的基因转录敲低（即，组织蛋白酶B，一种肠道定位的血红蛋白消化酶）;已知靶向组织蛋白酶B的常规RNAi递送可见的表型--抑制胚泡的生长。目的2采用目的1的逆转录病毒转基因方法，即基因“敲入”，但旨在建立可遗传的基因“敲除”。目的1和2将共同研究S. mansoni在公共卫生方面，这项研究旨在建立创新的方法来确定染色体基因的重要性，以帮助开发新的治疗和控制血吸虫病的疗法。

DESCRIPTION (provided by applicant): Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of parasite gene function and expression. This is the long term objective of our studies. We propose to modify the Moloney murine leukemia retroviral (MMLV) vector pLNHX to incorporate luciferase and other reporter genes under control of endogenous schistosome gene promoters. pLNHX constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) will be used to transfect GP2-293 cells to produce replication incompetent retrovirus particles pseudo-typed with VSVG. In Aim 1, the capacity of MMLV-VSVG retrovirus to transduce Schistosoma mansoni will be investigated. Developmental stages of schistosomes, including sporocysts, schistosomula and adults will be exposed to the retrovirus. Retroviral transduction of schistosomes will be facilitated by incubation with polybrene, phosphatidylserine and/or by centrifugation. The early stages of binding and uptake of virus to the tegument will be investigated by the immunofluorescence co-localization of VSVG and retroviral capsid proteins, and ultrastructural techniques. Downstream events, including integration of the pro-viral form of the retroviral transgene into schistosome chromosomes, transcription from integrated reporter genes, and activity of translated reporter proteins, will be investigated by Southern hybridization analysis, inverse PCR and related procedures, immunoblotting, and reporter proteins assays. In Aim 2, we will transduce schistosomes with MMLV-VSVG virions modified with a transgene cassette encoding gene-specific, double stranded RNA (rather than a reporter gene such as luciferase, as in Aim 1). We will investigate whether this transduction is heritable and whether it leads to knockdown of gene transcription of a model target gene (i.e., cathepsin B, a gut-localized, hemoglobin-digesting enzyme); conventional RNAi targeting cathepsin B is known to deliver a visible phenotype - stunting of growth of schistosomula. Aim 2 employs the retroviral transgenesis approach of Aim 1, i.e. gene "knock-in", but is designed to establish heritable gene "knock-out". Together, Aims 1 and 2 will investigate "knock-in, knock-out" transgenesis for S. mansoni. In terms of public health, this investigation seeks to establish innovative methods to determine the importance of schistosome genes to aid the development of new therapies to treat and control schistosomiasis.