目的 建立N-乙酰天冬氨酰谷氨酸(NAAG)肽酶基因剔除小鼠模型,为在体研究NAAG肽酶基因的生物学功能并揭示其在脑损伤后继发性脑损害进程中的所起的作用创造条件.方法 根据小鼠NAAG肽酶基因组的序列,设计基因剔除策略,构建基因剔除载体NAAG-KO-pBR322,以电穿孔方法将基因剔除载体导入胚胎干细胞(ES),应用G418和更昔洛韦进行正负筛选,获得双抗性克隆,聚合酶链式反应(PCR)鉴定并测序获得正确同源重组的ES细胞克隆.结果 同源重组的ES细胞注入小鼠囊胚后获得11只嵌合率＞50％嵌合体雄性小鼠,嵌合体小鼠与C57 BL/6J雌鼠交配后获得11只杂合子小鼠,其中雄性7只,雌性4只.在雌、雄杂合子小鼠交配的后代中获得7只纯合子小鼠,PCR鉴定其基因型,逆转录PCR (RT-PCR)提示该基因鼠未表达NAAG肽酶.结论 我们成功建立了NAAG肽酶基因剔除小鼠模型,其中纯合子小鼠未出现胚胎致死现象；初步的表型观察未发现NAAG肽酶基因剔除小鼠出现异常改变.%Objective To establish N-acetylaspartylglutamate (NAAG) peptidase gene knockout mouse model and to create the condition for farther in vivo study of its biological function and its role in the secondary brain damage after brain injury. Methods According to the NAAG peptidase genomic DNA sequence ,the strategy of gene targeting was established, and the gene knockout vector (NAAG-KO-pBR322) was constructed. Electroporation of embryonic stem (ES) cells with the gene knockout vector and screening of both G418 and Ganciclovir resistant clones were performed. The homologous recombined ES ceD clones were identified by polymerase chain reaction (PCR). Results After transplantation of homologous recombined ES cells into blastocysts through microinjection, there were 11 male chimeras bom with embedment rate >50%. The male chimeras then were bred with C57BL/6J female mice and 7(♂) and 4 (♀) offsprings with heterozygous genotype were acquired. Some heterozygote mice were intercrossed to generate mutant homozygotes. Finally, 7 mutant homozygote mice were obtained, whose genotype was identified by PCR. Reverse transcription PCR (RT-PCR) suggested that the NAAG peptidase gene was not expressed. Conclusion The NAAG peptidase gene knockout mice has been successfully established. The embryonic lethality in homologous mutant mice does not occur, and no abnormal change is found by preliminary observation of phenotype for NAAG peptidase gene knockout nice.
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