Establishment of stable high expression cell line with green fluorescent protein and resistance genes.
In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.
Zhang
Shengtao
S
Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Wenli
W
He
Peigen
P
Gong
Feili
F
Yang
Dongliang
D
D000818:Animals; D016466:CHO Cells; D002469:Cell Separation; D006224:Cricetinae; D003412:Cricetulus; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D000072597:Hepatitis A Virus Cellular Receptor 2; D019868:Kanamycin Kinase / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
C448908:HAVCR2 protein, human; D000072597:Hepatitis A Virus Cellular Receptor 2; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase