Department of Microbiology, Immunology & Tropical Medicine, The George Washington University Medical Center, Ross Hall 448, 2300 I Street NW, Washington, DC 20037, USA.
Transgenic Res. 2012 Jun;21(3):511-21. doi: 10.1007/s11248-011-9548-0. Epub 2011 Sep 28.
Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.
血吸（裂）虫或血吸虫是人类血吸虫病的病原体，是主要的被忽视热带病之一。已经报道了血吸虫的基因组草案序列，但需要功能基因组学工具来研究新报告基因的作用和必要性。基于载体的 RNA 干扰可以为血吸虫的功能基因组学分析做出贡献。我们使用编码报告萤火虫荧光素酶的 mRNA 作为模型靶标，比较了人类 U6 基因中的血吸虫和人类启动子在驱动人纤维肉瘤细胞和培养的血吸虫中的 shRNA 的性能。此外，还利用了逆转录病毒[鼠白血病病毒（MLV）]和质粒（piggyBac，pXL-Bac II）载体。血吸虫 U6 基因启动子长 270bp，人 U6 基因启动子长 264bp；它们有 41%的同源性。用假型 MLV 病毒粒子转导 HT1080 纤维肉瘤细胞和曼氏血吸虫的幼体后，用编码人 U6 启动子驱动的 shRNA 的病毒粒子观察到更强的荧光素酶活性降低，而用血吸虫 U6 启动子驱动的病毒粒子则观察到更强的荧光素酶活性降低。用 pXL-Bac-II 构建体转染 HT1080 细胞和幼体后也出现了类似的趋势-用编码人 U6 启动子的构建体观察到更强的荧光素酶活性降低，而用血吸虫 U6 启动子的构建体则观察到更强的荧光素酶活性降低。这些发现表明，人 U6 基因启动子驱动的 shRNA 活性比其血吸虫同源物更强，不仅在人癌细胞系中如此，在幼虫血吸虫中也是如此。这种 RNA 聚合酶 III 启动子代表了基于载体的 RNA 干扰研究在血吸虫和相关扁形动物寄生虫中的一个潜在有价值的组成部分。
