Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a...

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Autores principales:	Tatiana Flisikowska, Irmgard S Thorey, Sonja Offner, Francesca Ros, Valeria Lifke, Bryan Zeitler, Oswald Rottmann, Anna Vincent, Lei Zhang, Shirin Jenkins, Helmut Niersbach, Alexander J Kind, Philip D Gregory, Angelika E Schnieke, Josef Platzer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/28865b2f7cbb4665ba7b4622b6f2e4fd
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spelling	oai:doaj.org-article:28865b2f7cbb4665ba7b4622b6f2e4fd2021-11-18T06:52:11ZEfficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.1932-620310.1371/journal.pone.0021045https://doaj.org/article/28865b2f7cbb4665ba7b4622b6f2e4fd2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21695153/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.Tatiana FlisikowskaIrmgard S ThoreySonja OffnerFrancesca RosValeria LifkeBryan ZeitlerOswald RottmannAnna VincentLei ZhangShirin JenkinsHelmut NiersbachAlexander J KindPhilip D GregoryAngelika E SchniekeJosef PlatzerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 6, p e21045 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tatiana Flisikowska Irmgard S Thorey Sonja Offner Francesca Ros Valeria Lifke Bryan Zeitler Oswald Rottmann Anna Vincent Lei Zhang Shirin Jenkins Helmut Niersbach Alexander J Kind Philip D Gregory Angelika E Schnieke Josef Platzer Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
description	Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
format	article
author	Tatiana Flisikowska Irmgard S Thorey Sonja Offner Francesca Ros Valeria Lifke Bryan Zeitler Oswald Rottmann Anna Vincent Lei Zhang Shirin Jenkins Helmut Niersbach Alexander J Kind Philip D Gregory Angelika E Schnieke Josef Platzer
author_facet	Tatiana Flisikowska Irmgard S Thorey Sonja Offner Francesca Ros Valeria Lifke Bryan Zeitler Oswald Rottmann Anna Vincent Lei Zhang Shirin Jenkins Helmut Niersbach Alexander J Kind Philip D Gregory Angelika E Schnieke Josef Platzer
author_sort	Tatiana Flisikowska
title	Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
title_short	Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
title_full	Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
title_fullStr	Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
title_full_unstemmed	Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
title_sort	efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/28865b2f7cbb4665ba7b4622b6f2e4fd
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Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

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