title: Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat.
abstract: BACKGROUND
  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 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.
  
  
  RESULTS
  After 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institute of Laboratory Animal Science, Hannover Medical School, Carl-Neuberg-Str,1, 30625, Hannover, Germany. Zschemisch.Nils-Holger@mh-hannover.de
authors.forename: Nils-Holger
authors.initials: NH
authors.lastname: Zschemisch
authors.forename: Silke
authors.initials: S
authors.lastname: Glage
authors.forename: Dirk
authors.initials: D
authors.lastname: Wedekind
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Weinstein
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Cui
authors.forename: Martina
authors.initials: M
authors.lastname: Dorsch
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Hedrich
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D004622:Embryo, Mammalian / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D005434:Flow Cytometry; D016368:Frameshift Mutation / Q000235:genetics; D018390:Gene Targeting; D005838:Genotype; D005854:Germ Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008212:Lymphocyte Depletion; D008221:Lymphoid Tissue / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011917:Rats, Inbred Lew; D011922:Rats, Mutant Strains; D016335:Zinc Fingers
chemical_list: D018398:Homeodomain Proteins; C064658:RAG-1 protein; D004720:Endonucleases