TAFA2基因剔除小鼠模型的建立

目的 建立TAFA2基因剔除小鼠模型,为在体研究TAFA2基因的生物学功能及其与中枢神经系统发生、发育的关系创造条件. 方法 运用生物信息学手段确定小鼠TAFA2基因组的序列,设计基因剔除策略,构建基因剔除载体pBR322-MK-MCS-TAFA2,以电穿孔方法将基因剔除载体导入ES细胞,用G418和Ganciclovoir进行正负筛选,获得双抗性克隆,PCR鉴定出正确同源重组的ES细胞克隆. 结果 同源重组的ES细胞注入小鼠囊胚后获得嵌合体小鼠,嵌合体小鼠与C57BL/6J小鼠交配后获得Aguoti（野生型）毛色的小鼠41只,其中14只为TAFA2基因剔除杂合子小鼠,阳性率为34.1%.在雌、雄杂合子小鼠交配的后代中获得纯合子小鼠,初步的表型观察未发现TAFA2基因剔除小鼠出现异常改变. 结论 已成功建立了TAFA2基因剔除小鼠模型,其中纯合子小鼠未出现胚胎致死现象.

Generation of TAFA2 Gene Knockout Mouse Model

Objective To establish TAFA2 gene knockout mouse model and to create the condition for further in vivo study of its biological function and its role of development and growth in central nervous system. Methods The mouse genomic DNA sequence of TAFA2 gene was verified through bioinformatic analysis. According to the TAFA2 genomic DNA sequence, the strategy of gene targeting and constructing of knockout vector （pBR322-MK-MCS-TAFA2） were established. The knockout region spans from exon 4 to 5 of TAFA2 gene in mouse genome. The gene knockout vector pBR322- MK-MCS-TAFA2 was constructed and confirmed by restriction enzyme digestion and sequencing. Electroporation of ES cells with pBR322-MK-MCS-TAFA2 and screening of both G418 and Ganciclovoir resistant clones were performed according to common protocol. The homologous recornbined ES cell clones were identified by PCR. Results After transplantation of homologous recombined ES cells into blastocysts through microinjection and mating of chimeras with C57BL/6J mice, 41 offspring with Aguoti fur in color were acquired, 14 of them （34.1%） show heterozygous genotype for TAFA2. Some heterozygote mice were intercrossed to generate mutant homozygotes. Finally, the cohort of mutant homozygotes for TAFA2 were established. Any abnormal change was not found by preliminary observation of phenotype for heterozygous and homozygous mice. Conclusions The TAFA2 gene knockout mice have been established. The embryonic lethality in homologous mutant mice was not occurred.