Transduction of Schistosoma mansoni by pseudotyped retrovirus

假型逆转录病毒转导曼氏血吸虫

• 批准号: 7799460
• 负责人: Paul J Brindley
• 金额: $ 4.73万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-24 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7799460
• 关键词: Address; Adult; Amino Acids; Binding; Biological Assay; Biology; Capsid Proteins; Cathepsins; Cathepsins B; Cells; Centrifugation; Chromosomes; Cysteine Protease; DNA; Development; Digestion; Double-Stranded RNA; Electroporation; Enzymes; Event; Gene Expression; Gene Targeting; Gene Transfer Techniques; Genes; Genetic Transcription; Genome; Germ; Glycoproteins; Growth; Hemoglobin; Heritability; Immunoblotting; Immunofluorescence Immunologic; Inverse Polymerase Chain Reaction; Investigation; Knock-in Mouse; Knock-out; Light; Luciferases; Methods; Modeling; Molecular; Moloney Leukemia Virus; Mus; Nature; Organism; Parasite Control; Parasites; Pathway interactions; Peptide Hydrolases; Performance; Phenotype; Phosphatidylserines; Plasmids; Polybrene; Procedures; Proteins; Proteolysis; Proviruses; Public Health; RNA; RNA Interference; Reporter; Reporter Genes; Research; Research Personnel; Retroviral Vector; Retroviridae; Reverse Transcription; Schistosoma; Schistosoma mansoni; Schistosomiasis; Source; Sporocysts; Staging; Supplementation; System; Techniques; Testing; Transgenes; Transgenic Organisms; Translating; Vesicular stomatitis Indiana virus; Viral; Virion; Virus; base; design; design and construction; gene function; innovation; leukemia; murine retroviral vector; particle; programs; promoter; retroviral transduction; southern hybridization; uptake; vector

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.

描述（由申请人提供）：培养培养基的逆转录病毒转导提供了一种潜在的手段，以建立血吸虫的转基因线，从而促进寄生虫基因功能和表达的阐明。这是我们研究的长期目标。我们建议修改Moloney鼠白血病逆转录病毒（MMLV）载体PLNHX，以将荧光素酶和其他报告基因纳入内源性血吸虫基因启动子的控制。 PLNHX构建体和编码小囊炎病毒糖蛋白（VSVG）的质粒将用于转染GP2-293细胞，以产生无能力的逆转录病毒颗粒，以伪造用VSVG构型。在AIM 1中，将研究MMLV-VSVG逆转录病毒逆转曼氏菌的能力。血吸虫的发育阶段，包括孢子囊肿，血吸虫和成年人将暴露于逆转录病毒。通过与聚甲烯，磷脂酰丝氨酸和/或离心孵育，将促进血吸虫的逆转录。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，将研究结合和摄取病毒对TEGUMEMENT的早期阶段。下游事件，包括将逆转录病毒转基因的促病毒形式整合到螺螺旋体染色体中，综合报告基因的转录以及翻译报告基因蛋白的活性，将通过南方杂交分析，逆PCR及相关程序，免疫印象，免疫印象和报告蛋白的蛋白质进行研究。在AIM 2中，我们将用MMLV-VSVG病毒体转导通过编码基因特异性，双链RNA的转基因盒（而不是诸如Luciferase）的MMLV-VSVG病毒体（如AIM 1）。我们将研究这种转导是否可遗传，以及它是否导致模型靶基因的基因转录敲低（即，凝结蛋白酶B，一种肠道局部化的，血红蛋白消化酶）；已知靶向组织蛋白酶B的常规RNAi可以提供可见的表型 - 血吸虫生长的阻滞。 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2一起，将研究S. Mansoni的“敲门，敲除”转基因。在公共卫生方面，这项调查旨在建立创新的方法，以确定棘体基因在帮助开发新疗法以治疗和控制血吸虫病的重要性。