Furusawa Tadashi, Hosoe Misa, Ohkoshi Katsuhiro, Takahashi Seiya, Kiyokawa Nobutaka, Fujimoto Jun-ichiro, Amemiya Hiroshi, Suzuki Seiichi, Tokunaga Tomoyuki
Developmental Biology Department, National Institute of Agrobiological Sciences, Ikenodai 2, Kukisaki, Inashiki, Ibaraki 305-8602, Japan.
Mol Immunol. 2003 May;39(14):871-8. doi: 10.1016/s0161-5890(03)00008-7.
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.
为了培育用于异种移植的严重联合免疫缺陷（SCID）家畜，我们尝试在不进行基因敲除的情况下产生SCID表型。基于已报道的小鼠RAG1突变体，我们通过对催化结构域内的三个残基（D602A、D710A和E964A）进行诱变构建了相应的兔RAG1突变体。正如预期的那样，这些突变体在人工底物上均无催化活性，并抑制野生型RAG1的重组。此外，用增强型绿色荧光蛋白（EGFP）替换RAG1的N末端大大提高了蛋白质稳定性，并且三重突变体RAG1在体外抑制野生型活性的能力提高了两倍。我们培育了携带后一种突变体的转基因小鼠，以评估其对体内V（D）J重组的影响。七只转基因小鼠中有四只的血清IgM水平降至对照水平的约30 - 50%。我们的结果表明，无需基因敲除即可培育用于再生医学的免疫缺陷动物。
