Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of parasite gene function and expression. This is the long term objective of our studies. We propose to modify the Moloney murine leukemia retroviral (MMLV) vector pLNHX to incorporate luciferase and other reporter genes under control of endogenous schistosome gene promoters. pLNHX constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) will be used to transfect GP2-293 cells to produce replication incompetent retrovirus particles pseudo-typed with VSVG.
In Aim 1, the capacity of MMLV-VSVG retrovirus to transduce Schistosoma mansoni will be investigated. Developmental stages of schistosomes, including sporocysts, schistosomula and adults will be exposed to the retrovirus. Retroviral transduction of schistosomes will be facilitated by incubation with polybrene, phosphatidylserine and/or by centrifugation. The early stages of binding and uptake of virus to the tegument will be investigated by the immunofluorescence co-localization of VSVG and retroviral capsid proteins, and ultrastructural techniques. Downstream events, including integration of the pro-viral form of the retroviral transgene into schistosome chromosomes, transcription from integrated reporter genes, and activity of translated reporter proteins, will be investigated by Southern hybridization analysis, inverse PCR and related procedures, immunoblotting, and reporter proteins assays.
In Aim 2, we will transduce schistosomes with MMLV-VSVG virions modified with a transgene cassette encoding gene-specific, double stranded RNA (rather than a reporter gene such as luciferase, as in Aim 1). We will investigate whether this transduction is heritable and whether it leads to knockdown of gene transcription of a model target gene (i.e., cathepsin B, a gut-localized, hemoglobin-digesting enzyme);conventional RNAi targeting cathepsin B is known to deliver a visible phenotype - stunting of growth of schistosomula.
Aim 2 employs the retroviral transgenesis approach of Aim 1, i.e. gene """"""""knock-in"""""""", but is designed to establish heritable gene """"""""knock-out"""""""". Together, Aims 1 and 2 will investigate """"""""knock-in, knock-out"""""""" transgenesis for S. mansoni. In terms of public health, this investigation seeks to establish innovative methods to determine the importance of schistosome genes to aid the development of new therapies to treat and control schistosomiasis.
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