Transduction of Schistosoma mansoni by pseudotyped retrovirus

描述（由申请人提供）：培养血吸虫的逆转录病毒转导为建立血吸虫转基因系提供了一种潜在的手段，从而有助于阐明寄生虫基因的功能和表达。这是我们研究的长期目标。我们建议对Moloney小鼠白血病逆转录病毒（MMLV）载体pLNHX进行修饰，使其在内源性血吸虫基因启动子控制下加入荧光素酶和其他报告基因。将利用pLNHX构建体和编码水泡性口炎病毒糖蛋白（VSVG）的质粒转染GP2-293细胞，产生具有VSVG伪分型的复制能力不足的逆转录病毒颗粒。在Aim 1中，将研究MMLV-VSVG逆转录病毒转导曼氏血吸虫的能力。发育阶段的血吸虫，包括孢子囊、血吸虫和成虫都会接触到逆转录病毒。用聚苯乙烯、磷脂酰丝氨酸孵育和/或离心可促进血吸虫的逆转录病毒转导。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，将研究病毒与被膜结合和摄取的早期阶段。下游事件，包括逆转录病毒转基因的前病毒形式整合到血吸虫染色体中，整合报告基因的转录，翻译的报告蛋白的活性，将通过Southern杂交分析，反式PCR和相关程序，免疫印迹和报告蛋白分析进行研究。在aims 2中，我们将用转基因盒式编码基因特异性双链RNA修饰的MMLV-VSVG病毒粒子转导血吸虫（而不是像aims 1中那样使用荧光素酶等报告基因）。我们将研究这种转导是否可遗传，以及它是否会导致模式靶基因（即组织蛋白酶B，一种肠道定位的血红蛋白消化酶）的基因转录下调；以组织蛋白酶B为靶点的常规RNAi可导致血吸虫生长发育迟缓。Aim 2采用了Aim 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“敲出”。目标1和目标2将共同研究曼氏球菌的“敲入敲出”转基因。在公共卫生方面，这项调查旨在建立创新方法来确定血吸虫基因的重要性，以帮助开发治疗和控制血吸虫病的新疗法。

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.