CAJ | 학술논문

用电穿孔法将线性化的质粒pEGFP-N3分别导入来自129/ter、C57BL/6J和BALB/c 3个品系的小鼠胚胎干细胞MESPU-13、MESPU-35和MESPU-62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85%的被EGFP稳定标记的细胞株5个(129/ter 2个、C57BL/6J 1个、BALB/c 2个),分别命名为MESPU-13/G1和MESPU-13/G2、MESPU-35/G1、MESPU-62/G1和 MESPU-62/G2.从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征.
용전천공법장선성화적질립pEGFP-N3분별도입래자129/ter、C57BL/6J화BALB/c 3개품계적소서배태간세포MESPU-13、MESPU-35화MESPU-62중,경G418사선、형광현미경경검、양성극륭확증、류식세포의분선、재확증이급핵형분석등과정,분별득도핵형대우85%적피EGFP은정표기적세포주5개(129/ter 2개、C57BL/6J 1개、BALB/c 2개),분별명명위MESPU-13/G1화MESPU-13/G2、MESPU-35/G1、MESPU-62/G1화 MESPU-62/G2.종불동품계중각선일개증식생장쾌、형태전형적표기세포주,진행감성린산매염색、oct4기인산물적표체검측、류배형성화체내분화감정,결과표명소득도적핵형정상적、은정표기적ES세포계구유원ES세포적전형특정.
The linearized plasmid pEGFP-N3 was electroporated into three different mouse ES cell lines MESPU-13,MESPU-35 and MESPU-62 derived from 129/ter,C57BL/6J and BALB/c mouse strains respectively.Resistant clones were selected in the presence of G418 and then were identified under the fluorescence microscope through blue exciting light.Positive green clones were primarily expanded and further sorted using FACS(fluorescence activated cell sorter).Finally five EGFP stable integrated cell strains were obtained and were expanded (2 strains from 129/ter,1 strain from C57BL/6J and 2 strains from BALB/c).Each of the five cell strains presents high proliferation growth rate and typical morphology characters of ES cells and their colonies.More than 85% cells of each cell strain contain normal diploid karyotype.Then some analysis such as the AP (alkaline phosphatase) staining,oct4 gene expression assay,embryonic body formation and differentiated test in vivo and in vitro were made.The results indicated that the stable labeled ES cell strains had the normal karyotypes and maintained the ES cell typical characteristics.