Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes

Abstract

Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and S. mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modified to incorporate the human telomerase reverse transcriptase gene (hTERT) as a reporter, under the control of the retroviral long terminal repeat (LTR). Pseudotyped virions were employed to transduce S. japonicum to investigate the utility of retrovirus-mediated transgenesis of S. japonicum and the activity of human telomerase reverse transcriptase as a reporter transgene in schistosomes. Schistosomules perfused from experimentally infected rabbits were cultured for six days after exposure to the virions after which genomic DNAs from virus-exposed and control worms were extracted. Analysis of RNA from transduced parasites and immunohistochemistry of thin parasite sections revealed expression of hTERT in the transduced worms. Expression of hTERT was also confirmed by immunoblot analysis. These findings indicated that S. japonicum could be effectively transduced by VSVG pseudotyped retrovirus carrying the hTERT gene. Given the potential of hTERT to aid in derivation of immortalized cells, these findings suggest that this pantropic retroviral approach can be employed to transduce cells from specific tissues and organs of schistosomes to investigate the influence of transgene hTERT on growth and proliferation of schistosome cells.

Transgenesis inStrongyloidesand related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing

“…Studies in schistosomes identify the use of pseudotyped retroviral vectors as another approach to DNA transformation (Yang et al 2010; Rinaldi et al 2012; Hagen et al 2015). It is likely that the success of approach stems to a significant degree from the susceptibility of the schistosome tegument to invasion by the pseudotyped retroviruses and that the same caveats as discussed for electroporation will apply in the application of this approach to parasitic nematodes.…”

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…Both vector-based RNAi and expression of foreign transgenes have begun to be deployed in studies with schistosomes, with potential outcomes including validation of putative vaccine candidates and even the overexpression of candidate antigens. Pseudotyped murine leukemia virus and the piggyBac transposon have both been shown to transduce developmental stages of schistosomes, leading to chromosomal integration of retroviral transgenes and transgene reporter activity Morales et al, 2007;Yang et al, 2010). Moreover, a vector-based RNAi approach has been reported in which the MLV transgene encoded a long hairpin RNA specific for a schistosome protease involved in hemoglobinolysis (Tchoubrieva et al 2010).…”

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

“…We have shown that vesicular stomatitis virus glycoprotein pseudotyped murine leukemia virus (VSVG-MLV) virions can transduce different developmental stages of S. mansoni including adult worms, schistosomules and eggs, leading to integration of reporter proviral transgenes into schistosome chromosomes (Kines et al 2008; Kines et al 2010; Rinaldi et al 2011). Moreover, VSVG-MLV has recently shown utility in vector based RNA interference in adult schistosomes (Tchoubrieva et al 2010) and for expression of oncogenes in larval blood flukes (Yang et al 2010). Indeed, although other approaches are also in development for functional genomics of parasitic helminths (Dvorak et al 2010; Lok 2009; Morales et al 2007), pseudotyped MLV appears more well advanced at this time.…”