Kines Kristine J, Mann Victoria H, Morales Maria E, Shelby Bryan D, Kalinna Bernd H, Gobert Geoffrey N, Chirgwin Sharon R, Brindley Paul J
Department of Tropical Medicine and Center for Infectious Diseases, Tulane University, Health Sciences Center, New Orleans, LA 70112, USA.
Exp Parasitol. 2006 Apr;112(4):209-20. doi: 10.1016/j.exppara.2006.02.003. Epub 2006 Mar 10.
Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.
培养的血吸虫的逆转录病毒转导提供了一种建立血吸虫转基因品系的潜在方法，从而有助于阐明血吸虫基因的功能和表达。莫洛尼鼠白血病逆转录病毒（MMLV）载体pLNHX被修饰，以在来自剪接前导RNA和HSP70基因的血吸虫内源基因启动子的控制下整合EGFP或荧光素酶报告基因。这些构建体和编码水泡性口炎病毒糖蛋白（VSVG）的质粒与GP2-293细胞一起用于产生用VSVG包膜假型化的无复制能力的逆转录病毒颗粒。通过与聚凝胺孵育和/或通过离心促进曼氏血吸虫的几个发育阶段，包括子孢蚴，暴露于这些病毒粒子。通过VSVG包膜和逆转录病毒衣壳蛋白的免疫荧光共定位证明了病毒与寄生虫体表结合和摄取的早期阶段。Southern杂交分析表明MMLV构建体的前病毒形式整合在从暴露于病毒的血吸虫中分离的基因组DNA中。此外，对从病毒处理的寄生虫中分离的RNA的分析表明存在编码报告转基因的转录物。这些结果共同表明VSVG假型化的MMLV对培养的血吸虫进行了有效的转导，并为可遗传的血吸虫转基因提供了一条可行的途径。
