Pseudotyped murine leukemia virus for schistosome transgenesis: approaches, methods and perspectives
Pseudotyped murine leukemia virus for schistosome transgenesis: approaches, methods and perspectives
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DOI：
10.1007/s11248-013-9779-3
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发表时间：
2014-06-01
影响因子：
3
通讯作者：
Rinaldi, Gabriel
中科院分区：
文献类型：
--
作者：
Mann, Victoria H.;Suttiprapa, Sutas;Rinaldi, Gabriel
人类血吸虫、日本血吸虫、曼氏血吸虫和血吸虫的基因组序列草稿现已问世。血吸虫基因组包含类似于11,000个编码基因的蛋白质，其中很少有基因的功能被很好地理解。尽管如此，新描述的基因产物和新的非编码RNA代表了潜在的干预目标，正在开发分子工具来确定它们的重要性。在过去的十年里，在血吸虫基因操作工具的开发方面取得了值得注意的进展，包括RNAi对基因表达的干扰，以及由基因组整合合格载体介导的瞬时和稳定的转基因。逆转录病毒介导的转基因是一种已建立的模式物种功能基因组学方法。它提供了建立功能增减表型的方法，支持基于载体的RNA干扰，并代表了用于插入突变的强大的正向遗传学工具。以水泡性口炎病毒糖蛋白为假型的小鼠白血病病毒(MLV)介导了曼氏血吸虫的体细胞转化，并已证实整合的转基因基因在曼氏血吸虫中垂直传播，导致了转基因株系的建立。此外，编码抗生素耐药性的MLV转基因允许用适当的抗生素选择MLV转导的寄生虫。在这里，我们描述了生产和量化用于转导血吸虫发育阶段的假型MLV颗粒的详细方法。还介绍了分析MLV转导的血吸虫的方法，包括定量聚合酶链式反应和验证和定位转基因基因的基因组整合的高通量方法。我们预计，这些工具将在其他实验室的遗传学研究中找到用处，并用于其他被忽视的重要热带疾病的蠕虫病原体。
Draft genome sequences for the human schistosomes, Schistosoma japonicum, S. mansoni and S. haematobium are now available. The schistosome genome contains similar to 11,000 protein encoding genes for which the functions of few are well understood. Nonetheless, the newly described gene products and novel non-coding RNAs represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past decade, noteworthy advances has been reported towards development of tools for gene manipulation of schistosomes, including gene expression perturbation by RNAi, and transient and stable transfection including transgenesis mediated by genome integration competent vectors. Retrovirus-mediated transgenesis is an established functional genomic approach for model species. It offers the means to establish gain- or loss-of-function phenotypes, supports vector-based RNA interference, and represents a powerful forward genetics tool for insertional mutagenesis. Murine leukemia virus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein mediates somatic transgenesis in S. mansoni, and vertical transmission of integrated transgenes in S. mansoni has been demonstrated, leading the establishment of transgenic lines. In addition, MLV transgenes encoding antibiotic resistance allow the selection of MLV-transduced parasites with the appropriate antibiotics. Here we describe detailed methods to produce and quantify pseudotyped MLV particles for use in transducing developmental stages of schistosomes. Approaches to analyze MLV-transduced schistosomes, including qPCR and high throughput approaches to verify and map genome integration of transgenes are also presented. We anticipate these tools should find utility in genetic investigations in other laboratories and for other helminth pathogens of important neglected tropical diseases.