Antibiotic selection for schistosome transgenesis

描述（由申请人提供）：我们已经开发了一种利用鼠白血病病毒（MLV）来转染染色体卵的方法来获得转基因染色体。用来自MLV逆转录病毒转导卵的毛蚴感染蜗牛后，从蜗牛释放的尾蚴的基因组DNA揭示了转基因的存在，表明逆转录病毒转基因已通过无性发育周期传播，从而证实了种系转基因。转基因尾蚴经冷冻保存后，可保持对小鼠的感染性，并通过性发育（减数分裂）周期传递，产生F1代转基因尾蚴。来自暴露于MLV的多染色体群体的基因组DNA的高通量测序映射了转基因在整个基因组中的广泛和随机插入，沿着每个常染色体和性染色体，验证了该方法用于插入诱变的实用性。这些发现首次描述了染色体的大规模随机插入诱变和转基因在染色体中的种系传递。在这里，我们建议通过添加转基因染色体的抗生素选择来增强我们成功的染色体转基因方法。药物筛选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。转基因虫体的药物筛选将为转基因蠕虫的种群富集提供一种手段。最近，我们已经证明，MLV转导的表达新霉素耐药标记物的染色体可以在氨基糖苷类抗生素遗传霉素（G418）上被拯救。我们假设，基于这些初步的研究结果，可以建立转基因系的螺旋体转导受精卵内的螺旋体蛋壳，随后感染螺与由此产生的毛蚴使用抗生素的同时选择拯救转基因蠕虫和消除野生型蠕虫或转基因蠕虫不表达耐药性标记。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别抗生素的敏感性：（1）氨基糖苷类，如G418;（2）氨基糖苷类，如嘌呤霉素;（3）糖肽类，如博莱霉素-所有三种类别的选择性标记都很容易获得。目标2.抗生素抗性基因在逆转录病毒转导和转基因染色体中表达的优化。目标3。逆转录病毒转导的染色体发育阶段的抗生素选择，特别是在体外产卵，对这些抗生素。抗生素选择的可用性有望提高功能基因组学和对主要被忽视的热带疾病的蠕虫寄生虫的研究进展。

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.