Antibiotic selection for schistosome transgenesis

血吸虫转基因的抗生素选择

• 批准号: 8849840
• 负责人: Paul J Brindley
• 金额: $ 19.81万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-16 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8849840
• 关键词: Address; Aminoglycoside Antibiotics; Aminoglycosides; Aminonucleoside; Antibiotic Resistance; Antibiotics; Caenorhabditis elegans; Categories; Cell Line; Cells; Cestoda; Chromosome Pairing; Chromosomes; Codon Nucleotides; Combined Antibiotics; Communities; Derivation procedure; Development; Developmental Biology; Drug resistance; Elements; Gene Expression; Gene Transfer Techniques; Generations; Genes; Geneticin; Genome; Genomic DNA; Genomic approach; Genomics; Glycopeptides; Goals; Health; Helminths; High-Throughput Nucleotide Sequencing; Human; In Vitro; Insertional Mutagenesis; Insulator Elements; Intervention; Laboratories; Lead; Letters; Mammalian Cell; Maps; Meiosis; Methods; Molecular Genetics; Murine leukemia virus; Mus; Neomycin; Parasites; Parasitic Diseases; Parasitology; Performance; Pharmaceutical Preparations; Plants; Platyhelminths; Population; Puromycin; Puromycin Aminonucleoside; Reporting; Research; Resistance; Resource Sharing; Retroviral Vector; Retroviridae; Retroviridae Infections; Schistosoma; Schistosoma japonicum; Schistosoma mansoni; Schistosomatidae; Schistosomiasis; Sex Chromosomes; Snails; Staging; System; Testing; Tissues; Transgenes; Transgenic Organisms; Virion; Zeocin; antibiotic G 418; asexual; autosome; base; egg; foodborne; forward genetics; functional genomics; genetic selection; genome sequencing; improved; microbial; neglected tropical diseases; novel; novel strategies; parasite genome; pathogen; promoter; resistance gene; reverse genetics; tissue culture; tool; transmission process; vector; zygote

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.

描述（由申请人提供）：我们已经开发了一种方法来得出使用鼠白血病病毒（MLV）转染黑素卵的转基因血吸虫。在通过MLV逆转录病毒转导的卵子感染蜗牛，从蜗牛释放的cercariae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循环传播，从而通过无性发育循环传播。转基因尾cer虫可以冷冻保存并保留小鼠的感染性，然后通过性发育（减数分裂）周期传播，产生F1代的转基因血吸虫。从暴露于MLV映射的大量群体中的基因组DNA的高通量测序，并随机插入整个基因组的转基因，沿每个常染色体和性别染色体，验证了这种方法的插入性诱变方法的实用性。这些发现提供了首先描述染色体的大规模随机插入诱变以及在血吸虫中转基因的生殖线传播。在这里，我们建议通过添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中。转基因血块的药物选择将提供一种富集转基因蠕虫种群的方法。最近，我们证明了表达新霉素抗性标记物的MLV转导的血吸虫可以在氨基糖苷抗生素（G418）上营救。我们基于这些初步发现，假设可以通过转导阴茎蛋壳中的Zygote来确定血吸虫的转基因线，随后使用抗生素选择的奇迹选择抗生素来挽救转移性蠕虫和消除野生型蠕虫或跨性别的蠕虫，从而使蜗牛与产生的奇迹症感染了蜗牛。我们的三个具体目的是：目标1。研究曼森卵对三种离散类抗生素的敏感性：（1）氨基糖苷，例如G418; （2）氨基核苷，例如蛋白素; 3）糖肽，例如Zeocin-对于所有三个类别的可选标记都易于使用。 AIM 2。逆转录病毒转导和转基因线索中抗生素抗性基因表达的优化。 AIM 3。在这些抗生素上，逆转录病毒转导的黑素发育阶段的抗生素选择，尤其是体外产卵的卵。可以预期，抗生素选择的可用性可以增强功能性基因组学和研究进展，这些寄生虫负责主要被忽视的热带疾病。