Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo.
To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.
Furusawa
Tadashi
T
Developmental Biology Department, National Institute of Agrobiological Sciences, Ikenodai 2, Kukisaki, Inashiki, Ibaraki 305-8602, Japan.
Hosoe
Misa
M
Ohkoshi
Katsuhiro
K
Takahashi
Seiya
S
Kiyokawa
Nobutaka
N
Fujimoto
Jun-ichiro
J
Amemiya
Hiroshi
H
Suzuki
Seiichi
S
Tokunaga
Tomoyuki
T
D016475:3T3 Cells; D000361:Agammaglobulinemia / Q000097:blood / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D003062:Codon / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D015322:Gene Rearrangement, B-Lymphocyte; D016654:Genes, RAG-1; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D007075:Immunoglobulin M / Q000172:deficiency*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011817:Rabbits / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic; D016511:Severe Combined Immunodeficiency / Q000235:genetics* / Q000276:immunology; D014183:Transplantation, Heterologous; D045567:VDJ Recombinases
D003062:Codon; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007075:Immunoglobulin M; D008164:Luminescent Proteins; C499309:RAG2 protein, Oryctolagus cuniculus; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases