Generation of knockout mice for Nijmegen breakage syndrome gene, NBS1.

奈megen断裂综合征是一种常染色体隐性遗传病，其特征是联合免疫缺陷、易患癌症、小头畸形和生长迟缓。来自奈梅亨断裂综合征的细胞表现出染色体不稳定性、辐射敏感性和耐辐射DNA合成。构建Nbs1靶向载体，通过电穿孔法将其导入小鼠胚胎干细胞。选择G418抗性ES细胞克隆，通过Southern blot分析进行同源重组筛选。234个克隆中，3个克隆重组正确，用于胚泡注射。将嵌合小鼠与C57BL/6回交，得到F1小鼠。PCR分析证实了Nbs 1突变等位基因的种系传播。虽然Nbs1杂合小鼠是正常和可育的，但到目前为止还没有获得Nbs1阴性小鼠。结果表明，Nbs1蛋白的破坏可能导致胚胎致死，表明Nbs1蛋白复合物对胚胎存活至关重要。我们正在建立定时交配来研究胚胎致死的原因，并试图建立Nbs1无合子成纤维细胞来进一步确定Nbs1基因的功能。

Nijmegen breakage syndrome is an autosomal recessive disorder characterized by combined immunodeficiency, predisposition to cancer, microcephaly, and growth retardation. Cells from Nijmegen breakage syndrome display chromosomal instability, radiation sensitivity, and radioresistant DNA synthesis. An Nbs1 targeting vector was constructed and was introduced into mouse ES cells by electroporation. G418 resistant ES cell clones were selected and were screened for homologous recombination by Southern blot analysis. Out of 234 clones, 3 clones showed correct recombination and were used for blastocyst injection. Chimeric mice were back crossed with C57BL/6, and F1 mice were generated. PCR analysis demonstrated germ line transmission of the Nbs 1 mutant allele. Although the Nbs1 heterozygous mice were normal and fertile, Nbs1 null mice could not be obtained so far. The result suggested that the disruption of Nbs1 could cause embryonic lethality, indicating that Nbs1 protein complex is essential for viability. We are setting up timed matings to investigate the cause of embryonic lethality, and also trying to establish Nbs1 nullizygous fibroblasts to define further function of the Nbs1 gene.