Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.

Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseu...

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Main Authors: Gabriel Rinaldi, Sabine E Eckert, Isheng J Tsai, Sutas Suttiprapa, Kristine J Kines, José F Tort, Victoria H Mann, Daniel J Turner, Matthew Berriman, Paul J Brindley
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS Pathogens
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22911241/pdf/?tool=EBI
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spelling doaj-420675da718e475b97585bd56cfbe2f52021-04-21T17:09:56ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742012-01-0187e100282010.1371/journal.ppat.1002820Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.Gabriel RinaldiSabine E EckertIsheng J TsaiSutas SuttiprapaKristine J KinesJosé F TortVictoria H MannDaniel J TurnerMatthew BerrimanPaul J BrindleyFunctional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. DATABASE ACCESSION: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22911241/pdf/?tool=EBI
collection DOAJ
language English
format Article
sources DOAJ
author Gabriel Rinaldi
Sabine E Eckert
Isheng J Tsai
Sutas Suttiprapa
Kristine J Kines
José F Tort
Victoria H Mann
Daniel J Turner
Matthew Berriman
Paul J Brindley
spellingShingle Gabriel Rinaldi
Sabine E Eckert
Isheng J Tsai
Sutas Suttiprapa
Kristine J Kines
José F Tort
Victoria H Mann
Daniel J Turner
Matthew Berriman
Paul J Brindley
Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
PLoS Pathogens
author_facet Gabriel Rinaldi
Sabine E Eckert
Isheng J Tsai
Sutas Suttiprapa
Kristine J Kines
José F Tort
Victoria H Mann
Daniel J Turner
Matthew Berriman
Paul J Brindley
author_sort Gabriel Rinaldi
title Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
title_short Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
title_full Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
title_fullStr Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
title_full_unstemmed Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.
title_sort germline transgenesis and insertional mutagenesis in schistosoma mansoni mediated by murine leukemia virus.
publisher Public Library of Science (PLoS)
series PLoS Pathogens
issn 1553-7366
1553-7374
publishDate 2012-01-01
description Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. DATABASE ACCESSION: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.
url https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22911241/pdf/?tool=EBI
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