Progress with schistosome transgenesis

Abstract

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.

Fig. 1

Fig. 1A: schematic representation of the structure and mechanism of transposition of transposons. The terminal inverted repeats (TIR) (black arrows) contain binding sites for the transposase (white arrows). The element contains a single gene encoding the transposase. The NH2-terminal part of the transposase contains a DNA binding domain, followed by a nuclear localization signal. The COOH-terminal part of the protein is responsible for catalysis, including the DNA cleavage and rejoining reactions (not shown); B: schematic of the donor cassette in which the transposase open reading frame has been replaced with a reporter transgene driven by an endogenous promoter; C: cut and paste mechanism of transposition. The transposase initiates the excision of the transposon donor cassette with staggered cuts and reintegrates it at the target integration site; D: the single-stranded gaps at the integration site as well as the double-strand DNA breaks in the donor DNA are repaired by the host DNA repair machinery. After repair, the target is regenerated at the integration site in the host cell chromosome and at the site of excision from the donor plasmid [adapted from Handler (2002), Miskey et al. (2005), Morales et al. (2007) and Mann et al. (2008)].

Fig. 2

southern hybridization analysis of piggyBac transposon integration into schistosome chromosomes. A: representation of the (BssS 1)-linearized pXL-BacII-SmAct-Luc transposon construct. The transposon cassette included the firefly luciferase reporter (gray arrow) followed by the SV40 polyadenylation site (black box), driven by the schistosome actin gene promoter (black arrow) and flanked by the piggyBac terminal inverted repeats (white arrows); B: the structure of a piggyBac-mediated integration site, depicting variable length fragments expected following Sph I digestion of gDNA from piggyBac-transformed schistosomules. The luciferase probe (LUC) is indicated by a black bar; C: ethidium-stained gel of gDNA from Schistosoma mansoni digested with Sph I (left), southern hybridization of Sph I digested DNA (gDNA) to the labeled LUC probe (right), size standards in kilobases (kb) (Lane 1), gDNA from control schistosomes (Lane 2), gDNA from schistosomules after electroporation with donor piggyBac plasmid plus in vitro-transcribed transposase mRNA (Lane 3), gDNA from schistosomules seven days after electroporation with donor piggyBac plasmid alone (Lane 4). [From Morales et al. (2007), with permission].

Fig. 3

schematic representation of retroviral replication and integration into a schistosome cell. After entering the cell, the retrovirus RNA genome is reverse transcribed into double-stranded DNA by reverse transcriptase (RT) present in the virion. The DNA copy migrates to the cell nucleus and integrates into the host genome as the provirus. Viral mRNAs are transcribed from proviral DNA by host cell enzymes in the nucleus. Both spliced and unspliced mRNAs are translated into viral proteins in the cytoplasm. The capsid precursor protein, Gag, and RT are translated from full-length RNA. The glycoproteins are translated from spliced mRNA and transported to the cell plasma membrane. Immature virions containing Gag, RT and the genome RNA assemble near the modified cell membrane. [Adapted from Strauss and Strauss (2002)].