Kines Kristine J, Morales Maria E, Mann Victoria H, Gobert Geoffrey N, Brindley Paul J
Department of Tropical Medicine, and Biomedical Sciences Program, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
FASEB J. 2008 Aug;22(8):2936-48. doi: 10.1096/fj.08-108308. Epub 2008 Apr 10.
The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SR1. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes.
最近公布的两种主要人体血吸虫的基因组序列草图凸显了为这些重要病原体开发功能基因组学方法的迫切需求。该序列信息也使对转基因整合到血吸虫染色体中的全基因组规模研究变得可行。逆转录病毒介导的转导提供了一种建立血吸虫转基因系、阐明血吸虫基因功能和表达以及推进针对这些寄生虫的功能基因组学方法的手段。我们研究了用泡状口炎病毒糖蛋白（VSVG）假型化的莫洛尼鼠白血病病毒（MLV）用于曼氏血吸虫转导以及将报告转基因递送至血吸虫染色体的效用。将血吸虫童虫暴露于VSVG假型化的MLV病毒粒子，之后从转导的血吸虫中提取基因组DNA。Southern杂交分析表明在转导的血吸虫中存在前病毒MLV逆转录病毒。使用基于锚定PCR的方法回收的MLV转基因片段和侧翼血吸虫序列明确证明血吸虫染色体发生了体细胞转基因作用，而且揭示了逆转录病毒广泛整合到血吸虫染色体中。更具体地说，MLV转基因已插入到编码亲免素、锌指蛋白Sma-Zic等的基因附近，以及内源性血吸虫逆转座子fugitive和SR1附近。MLV转基因的前病毒整合似乎表现出一级序列位点特异性，靶向类似gGATcc的基序。由血吸虫肌动蛋白基因启动子驱动的报告荧光素酶转基因活性在转导的血吸虫童虫和成虫组织中表达。荧光素酶活性似乎在血吸虫童虫中呈发育性表达，培养1至2周后活性增加。这些发现表明VSVG假型化的MLV对曼氏血吸虫转基因作用的效用，预示着朝着寄生蠕虫的种系转基因作用和功能基因组学发展的一条可行途径，并为比较分子发病机制研究提供了基础，该研究涉及与血吸虫染色体相比逆转录病毒整合到人类染色体中所产生的染色体损伤。
