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

奈梅亨断裂综合征是一种常染色体隐性遗传疾病，其特征是联合免疫缺陷，易患癌症，小头畸形和生长迟缓。奈梅亨断裂综合征的细胞显示染色体不稳定性、辐射敏感性和抗辐射DNA合成。构建了Nbs 1靶向载体，并通过电穿孔将其导入小鼠ES细胞。选择G418抗性ES细胞克隆，并通过Southern印迹分析筛选同源重组。在234个克隆中，3个克隆显示正确的重组并用于囊胚注射。将嵌合小鼠与C57 BL/6回交，并产生F1小鼠。PCR分析表明Nbs 1突变等位基因的生殖系传播。虽然Nbs 1杂合子小鼠是正常的和可生育的，但迄今为止还不能获得Nbs 1敲除小鼠。结果表明，Nbs 1蛋白复合物的破坏可导致胚胎死亡，说明Nbs 1蛋白复合物对胚胎的存活至关重要。我们正在建立定时交配，以调查胚胎致死的原因，并试图建立Nbs 1缺失的成纤维细胞，以确定Nbs 1基因的进一步功能。

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.