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Updated: Jul 12, 2026

Molecular Evolution of the Tre Recombinase
Published on: May 29, 2008
Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo
Tadashi Furusawa1, Misa Hosoe, Katsuhiro Ohkoshi
1Developmental Biology Department, National Institute of Agrobiological Sciences, Ikenodai 2, Kukisaki, Inashiki, Ibaraki 305-8602, Japan.
Abstract:
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.
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