Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat
<p>Abstract</p> <p>Background</p> <p>Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronucle...
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| Format: | Article |
| Language: | English |
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BMC
2012-11-01
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| Series: | BMC Immunology |
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| Online Access: | http://www.biomedcentral.com/1471-2172/13/60 |
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| author | Zschemisch Nils-Holger Glage Silke Wedekind Dirk Weinstein Edward J Cui Xiaoxia Dorsch Martina Hedrich Hans-Jürgen |
| author_facet | Zschemisch Nils-Holger Glage Silke Wedekind Dirk Weinstein Edward J Cui Xiaoxia Dorsch Martina Hedrich Hans-Jürgen |
| author_sort | Zschemisch Nils-Holger |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat <it>Rag1</it> gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.</p> <p>Results</p> <p>After microinjection of <it>Rag1</it> specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated <it>Rag1</it> protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the <it>Rag1</it> protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4<sup>+</sup>/CD3<sup>+</sup>/TCRαβ<sup>+</sup> as well as CD8<sup>+</sup>/CD3<sup>+</sup>/TCRαβ<sup>+</sup> positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in <it>Rag1</it> mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient <it>Rag1</it> mutant rat.</p> <p>Conclusion</p> <p>The <it>Rag1</it> mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.</p> |
| first_indexed | 2024-12-12T22:49:34Z |
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| institution | Directory Open Access Journal |
| issn | 1471-2172 |
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| publishDate | 2012-11-01 |
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| spelling | doaj.art-f0a07a48faba4c599a378aa416a3e2da2022-12-22T00:09:07ZengBMCBMC Immunology1471-21722012-11-011316010.1186/1471-2172-13-60Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm ratZschemisch Nils-HolgerGlage SilkeWedekind DirkWeinstein Edward JCui XiaoxiaDorsch MartinaHedrich Hans-Jürgen<p>Abstract</p> <p>Background</p> <p>Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat <it>Rag1</it> gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.</p> <p>Results</p> <p>After microinjection of <it>Rag1</it> specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated <it>Rag1</it> protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the <it>Rag1</it> protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4<sup>+</sup>/CD3<sup>+</sup>/TCRαβ<sup>+</sup> as well as CD8<sup>+</sup>/CD3<sup>+</sup>/TCRαβ<sup>+</sup> positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in <it>Rag1</it> mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient <it>Rag1</it> mutant rat.</p> <p>Conclusion</p> <p>The <it>Rag1</it> mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.</p>http://www.biomedcentral.com/1471-2172/13/60<it>Rag1</it>Zinc-finger nucleasesRatLymphocytesNatural killer cellsHypoplastic thymus |
| spellingShingle | Zschemisch Nils-Holger Glage Silke Wedekind Dirk Weinstein Edward J Cui Xiaoxia Dorsch Martina Hedrich Hans-Jürgen Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat BMC Immunology <it>Rag1</it> Zinc-finger nucleases Rat Lymphocytes Natural killer cells Hypoplastic thymus |
| title | Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat |
| title_full | Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat |
| title_fullStr | Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat |
| title_full_unstemmed | Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat |
| title_short | Zinc-finger nuclease mediated disruption of <it>Rag1</it> in the LEW/Ztm rat |
| title_sort | zinc finger nuclease mediated disruption of it rag1 it in the lew ztm rat |
| topic | <it>Rag1</it> Zinc-finger nucleases Rat Lymphocytes Natural killer cells Hypoplastic thymus |
| url | http://www.biomedcentral.com/1471-2172/13/60 |
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