Antibiotic selection for schistosome transgenesis

描述（由申请人提供）：我们开发了一种利用小鼠白血病病毒（MLV）转染血吸虫卵获得转基因血吸虫的方法。用MLV逆转录病毒转导的卵中的miracidia感染蜗牛后，从蜗牛释放的尾蚴基因组DNA显示存在转基因，表明逆转录病毒转基因通过无性发育周期传播，从而证实了种系转基因。转基因尾蚴可以低温保存并保留对小鼠的传染性，并通过性发育（减数分裂）周期传播，产生F1代转基因血吸虫。对暴露于MLV的血吸虫种群的基因组DNA进行高通量测序，绘制出沿每个常染色体和性染色体在整个基因组中广泛和随机插入转基因的图谱，验证了该方法在插入诱变中的实用性。这些发现首次描述了大规模的、随机插入的染色体突变和一种转基因在血吸虫中的种系传播。在此，我们建议通过增加转基因血吸虫的抗生素选择来增强我们成功的血吸虫转基因方法。药物选择广泛应用于微生物病原体、哺乳动物细胞和植物细胞系的转基因研究。转基因血吸虫的药物选择为转基因血吸虫种群的丰富提供了途径。最近，我们已经证明，表达新霉素耐药标记的mlv转导的血吸虫可以在氨基糖苷类抗生素genticin （G418）上获救。基于这些初步研究结果，我们推测，可以通过在血吸虫蛋壳内转导合子，然后用由此产生的miracidia感染蜗牛，同时使用抗生素选择来拯救转基因蠕虫，并消除野生型蠕虫或不表达耐药maker的转基因蠕虫，从而建立转基因血吸虫系。我们的三个具体目标是：目标1。研究曼氏血吸虫卵对三种不同类型抗生素的敏感性：(1)氨基糖苷类，如G418；(2)氨基核苷，如嘌呤霉素；3)糖肽类，如zeocin——这三种类型的可选标记物都很容易获得。目标2。逆转录病毒转导和转基因血吸虫中抗生素耐药基因表达的优化。目标3。逆转录病毒转导的血吸虫发育阶段的抗生素选择，特别是在体外产卵时，对这些抗生素的影响。抗生素选择的可用性有望促进功能基因组学和对主要被忽视的热带病的寄生虫的研究进展。

DESCRIPTION (provided by applicant): We have developed a method to derive transgenic schistosomes that utilizes the murine leukemia virus (MLV) to transfect schistosome eggs. After infecting snails with miracidia from eggs transduced by the MLV retrovirus, genomic DNA from cercariae released from the snails revealed the presence of transgenes, demonstrating that retroviral transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. Transgenic cercariae could be cryopreserved and retained infectivity for mice, and were in turn transmitted through the sexual developmental (meiosis) cycle to produce F1 generations of transgenic schistosomes. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. These findings provided the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Here we propose to enhance our successful approach to schistosome transgenesis by the addition of antibiotic selection of transgenic schistosomes. Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would provide a means to enrich for populations of transgenic worms. Recently we have demonstrated that MLV-transduced schistosomules expressing a neomycin resistance marker could be rescued on the aminoglycoside antibiotic, geneticin (G418). We hypothesize, based on these preliminary findings, that transgenic lines of schistosomes can be established by transducing the zygote within the schistosome eggshell and subsequently infecting snails with the resulting miracidia using concurrent selection on antibiotics to rescue transgenic worms and eliminating wild type worms or transgenic worms not expressing the drug resistance maker. Our three specific aims are: Aim 1. Investigate sensitivity of Schistosoma mansoni eggs to three discrete classes of antibiotics: (1) aminoglycosides e.g. G418; (2) aminonucleosides e.g. puromycin; 3) glycopeptides e.g. zeocin - for which selectable markers for all three categories are readily available. Aim 2. Optimization of antibiotic resistance gene expression in retrovirus-transduced and transgenic schistosomes. Aim 3. Antibiotic selection of retrovirus-transduced schistosome developmental stages, in particular in vitro laid eggs, on these antibiotics. The availability of antibiotic selection can be expected to enhance functional genomics and research progress on helminth parasites responsible for major neglected tropical diseases.