Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

Abstract

BackgroundEngineered 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 Rag1 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.ResultsAfter microinjection of Rag1 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 Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 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 Rag1 mutant rat.ConclusionThe Rag1 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.

Genetic mutation of recombination activating gene 1 in Dahl salt-sensitive rats attenuates hypertension and renal damage

“…There were no other developmental abnormalities noted in these animals, although the potential impact of developmental changes and/or alteration in other pathways in the absence of Rag1 protein is a limitation of this gene deletion approach. Qualitatively, the phenotypes observed in the present study are similar to those previously reported in Rag1-/-mice (18) and recently reported in the LEW/Ztm rat with ZFN-mediated disruption of Rag1 (29).…”

Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency

“…The latter have a small, but nevertheless detectable, thymus (6) whereas three of the four mutant pigs lacked a thymus completely and the fourth had one that was extremely underdeveloped, as was also observed in pigs lacking an intact IL2RG gene (15,16). The lack of B and T cells is analogous to mouse, rat, and rare human RAG mutants (6,30,31), but, as with the porcine IL2RG knock-outs and human patients, RAG2…”

Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

“…Moreover, for mammalian species other than the mouse, genetic engineering was simply not possible due to an inability to derive legitimate ES cells despite considerable effort over many years. Recent advances in site-specific nuclease technologies (e.g ZFN, TALEN, CRISPR/Cas) are enabling direct, targeted deletion and targeted, sequential gene modifications via pronuclear injection of mouse embryos 10 and other species, including rat 11,12 . For genetic engineering, these technologies circumvent the need for ES cells.…”