Transduction of Schistosoma mansoni by pseudotyped retrovirus

描述(申请人提供)：用逆转录病毒转导培养的血吸虫为建立血吸虫转基因品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。这是我们研究的长远目标。我们建议对Moloney小鼠白血病逆转录病毒(MMLV)载体pLNHX进行修饰，以在内源性血吸虫基因启动子的控制下整合荧光素酶和其他报告基因。PLNHX构建物和水泡性口炎病毒糖蛋白(VSVG)编码质粒将被用来转染GP2-293细胞，以产生VSVG假型的复制不能复制的逆转录病毒颗粒。目的1研究MMLV-VSVG逆转录病毒转导曼氏血吸虫的能力。血吸虫的发育阶段，包括孢子囊、血吸虫幼虫和成虫将暴露在逆转录病毒中。通过与聚乙二烯、磷脂酰丝氨酸孵育和/或离心法，可以促进血吸虫的逆转录病毒转导。病毒与被膜结合和吸收的早期阶段将通过VSVG和逆转录病毒衣壳蛋白的免疫荧光共定位以及超微结构技术来研究。下游事件，包括反转录病毒转基因的亲病毒形式整合到血吸虫染色体，从整合的报告基因转录，以及翻译的报告蛋白的活性，将通过Southern杂交分析，反向聚合酶链式反应和相关程序，免疫印迹和报告蛋白分析进行研究。在目标2中，我们将用编码基因特异性双链RNA(而不是目标1中的荧光素酶等报告基因)的转基因盒修饰MMLV-VSVG病毒粒子来转导血吸虫。我们将调查这种转导是否可遗传，以及它是否导致模型靶基因(即组织蛋白B，一种肠道定位的血红蛋白消化酶)的基因转录下调；已知针对组织蛋白B的常规RNAi可带来可见的表型-血吸虫生长迟缓。AIM 2采用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“敲除”。AIMS 1和AIMS 2将共同调查曼氏葡萄球菌的“敲入、敲除”转基因。在公共卫生方面，这项调查试图建立创新的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.