Establishment of stable high expression cell line with green fluorescent protein and resistance genes
| Autor: | Feili Gong, Sheng-tao Zhang, Peigen He, Dongliang Yang, Wenli Liu |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
viruses
Recombinant Fusion Proteins Genetic Vectors Green Fluorescent Proteins Biomedical Engineering CHO Cells Cell Separation Biology Transfection Biochemistry law.invention Flow cytometry Green fluorescent protein Biomaterials chemistry.chemical_compound Cricetulus law Cricetinae Genetics medicine Animals Hepatitis A Virus Cellular Receptor 2 Earth-Surface Processes Growth medium medicine.diagnostic_test Kanamycin Kinase Chinese hamster ovary cell fungi Membrane Proteins Flow Cytometry Molecular biology Cell biology Eukaryotic Cells chemistry Cell culture Lipofectamine embryonic structures Recombinant DNA Plasmids |
| Zdroj: | Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban. 26(3) |
| ISSN: | 1672-0733 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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