Department of Microbiology, Immunology & Tropical Medicine, School of Medicine & Health Sciences, The George Washington University, Washington, DC, United States of America.
PLoS Pathog. 2012;8(7):e1002820. doi: 10.1371/journal.ppat.1002820. Epub 2012 Jul 26.
Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. 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. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. DATABASE ACCESSION: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.
功能研究将有助于描述人类血吸虫曼氏血吸虫、日本血吸虫和埃及血吸虫新获得的基因组序列的作用和必要性。为了将转基因作为这些病原体的一种功能方法来发展，我们之前证明了假型鼠白血病病毒 (MLV) 可以转导血吸虫，导致报告基因和短发夹 RNA 盒的染色体整合。在这里，我们研究了通过曼氏血吸虫发育周期传递转基因，方法是将转基因引入卵中。尽管 MLV 感染来自鼠肝的血吸虫卵在感染蜗牛方面非常有效，但在体外产卵（IVLE）中获得的尾蚴中检测到的转基因拷贝数高出 10 倍以上。用 MLV 转导的卵中的 miracidia 感染蜗牛后，从蜗牛释放的尾蚴的基因组 DNA 测序也揭示了转基因的存在，证明转基因已通过无性发育周期传递，从而确认了种系转基因。暴露于 MLV 的血吸虫种群的基因组 DNA 高通量测序将转基因广泛且随机地插入整个基因组，沿着每条常染色体和性染色体，验证了这种方法用于插入诱变的有效性。此外，种系传递的新霉素磷酸转移酶转基因拯救了培养的血吸虫幼虫免受抗生素 G418 的毒性，并且对在小鼠中进行有性繁殖的转基因蠕虫产生的卵进行的 PCR 分析证实了逆转录病毒转基因已传递给下一代（F1）。这些发现首次描述了染色体的大规模、随机插入诱变和血吸虫种系转基因的传递。表达抗生素抗性的转基因血吸虫系可以推进这些重要人类病原体的功能基因组学。数据库访问：本研究的序列数据已提交给欧洲核苷酸档案库（http://www.ebi.ac.uk/embl），登录号为 ERP000379。
