Labeling of three different mouse ES cell lines with the green fluorescent protein.
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.
Zhao
Wen-Ning
WN
College of Life Science, Peking University, Beijing 100871, China. zhouym@pku.edu.cn
Meng
Guo-Liang
GL
Xue
You-Fang
YF
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007621:Karyotyping; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D050814:Octamer Transcription Factor-3; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection / Q000379:methods
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase