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:	Rinaldi, Gabriel, Eckert, Sabine E., Tsai, Isheng J., Suttiprapa, Sutas, Kines, Kristine J., Tort, José F., Mann, Victoria H., Turner, Daniel J., Berriman, Matthew, Brindley, Paul J.
Format:	Online
Language:	English
Published:	Public Library of Science 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406096/

id	pubmed-3406096
recordtype	oai_dc
spelling	pubmed-34060962012-07-30 Germline Transgenesis and Insertional Mutagenesis in Schistosoma mansoni Mediated by Murine Leukemia Virus Rinaldi, Gabriel Eckert, Sabine E. Tsai, Isheng J. Suttiprapa, Sutas Kines, Kristine J. Tort, José F. Mann, Victoria H. Turner, Daniel J. Berriman, Matthew Brindley, Paul J. Research Article 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. Public Library of Science 2012-07-26 /pmc/articles/PMC3406096/ /pubmed/22911241 http://dx.doi.org/10.1371/journal.ppat.1002820 Text en Rinaldi et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Rinaldi, Gabriel Eckert, Sabine E. Tsai, Isheng J. Suttiprapa, Sutas Kines, Kristine J. Tort, José F. Mann, Victoria H. Turner, Daniel J. Berriman, Matthew Brindley, Paul J.
spellingShingle	Rinaldi, Gabriel Eckert, Sabine E. Tsai, Isheng J. Suttiprapa, Sutas Kines, Kristine J. Tort, José F. Mann, Victoria H. Turner, Daniel J. Berriman, Matthew Brindley, Paul J. Germline Transgenesis and Insertional Mutagenesis in Schistosoma mansoni Mediated by Murine Leukemia Virus
author_facet	Rinaldi, Gabriel Eckert, Sabine E. Tsai, Isheng J. Suttiprapa, Sutas Kines, Kristine J. Tort, José F. Mann, Victoria H. Turner, Daniel J. Berriman, Matthew Brindley, Paul J.
author_sort	Rinaldi, Gabriel
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
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.
publisher	Public Library of Science
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406096/
_version_	1611545874798739456

Germline Transgenesis and Insertional Mutagenesis in Schistosoma mansoni Mediated by Murine Leukemia Virus

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