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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 a mouse model of NAAG peptidase gene knockout mice in order to study the biological function of NAAG peptidase gene in vivo and to reveal its role in the process of secondary brain damage following brain injury The role of play to create conditions. Methods According to the sequence of mouse NAAG peptidase, gene knockout strategy was designed to construct gene knockout vector NAAG-KO-pBR322. The gene knockout vector was introduced into embryonic stem cells (ES) by electroporation, and G418 and ganciclovir Negative selection, double resistant clones obtained, identified by PCR and sequenced to obtain the correct homologous recombination of ES cell clones. Results Eleven chimeric male mice with chimerism rate> 50% were obtained after injection of homologous recombined ES cells into mouse blastocysts, and 11 heterozygous mice were obtained after mating with chimeric mice and C57BL / 6J females 7 males and 4 females. Seven homozygous mice were obtained from the offspring of female and male heterozygous mice, and their genotypes were identified by PCR. RT-PCR indicated that NAAG peptidase was not expressed in the mice. Conclusion We successfully established NAAG peptidase gene knockout mice model, in which homozygous mice did not appear embryonic lethality; preliminary phenotype observation did not find NAAG peptidase gene knockout mice abnormal changes.