Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering

ABSTRACT Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombina...

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Autores principales:	Sumiti Vinayak, Carrie F. Brooks, Anatoli Naumov, Elena S. Suvorova, Michael W. White, Boris Striepen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/98956d71a29d45efb132e73072f47b57
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spelling	oai:doaj.org-article:98956d71a29d45efb132e73072f47b572021-11-15T15:47:03ZGenetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering10.1128/mBio.02021-142150-7511https://doaj.org/article/98956d71a29d45efb132e73072f47b572014-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02021-14https://doaj.org/toc/2150-7511ABSTRACT Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite’s genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen. IMPORTANCE Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.Sumiti VinayakCarrie F. BrooksAnatoli NaumovElena S. SuvorovaMichael W. WhiteBoris StriepenAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 6 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Sumiti Vinayak Carrie F. Brooks Anatoli Naumov Elena S. Suvorova Michael W. White Boris Striepen Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
description	ABSTRACT Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite’s genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen. IMPORTANCE Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.
format	article
author	Sumiti Vinayak Carrie F. Brooks Anatoli Naumov Elena S. Suvorova Michael W. White Boris Striepen
author_facet	Sumiti Vinayak Carrie F. Brooks Anatoli Naumov Elena S. Suvorova Michael W. White Boris Striepen
author_sort	Sumiti Vinayak
title	Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
title_short	Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
title_full	Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
title_fullStr	Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
title_full_unstemmed	Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering
title_sort	genetic manipulation of the <named-content content-type="genus-species">toxoplasma gondii</named-content> genome by fosmid recombineering
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/98956d71a29d45efb132e73072f47b57
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Genetic Manipulation of the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Genome by Fosmid Recombineering

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