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[Expression of lipofect AMINE mediated human GM-CSF eukaryotic expressing vector in HFCL cells]
1Department of Hematology & Oncology, The First Clinical College, Norman Bethune University of Medical Sciences, Changchun 130021, China.
Objective:
To construct an eukaryotic expressing vector-pIRES1neo/hGM-CSF and express it in human bone marrow stromal cell line HFCL.
Methods:
Human granulocyte/macrophage colony-stimulating factor cDNA (hGM-CSF cDNA, 751 bp) was inserted into an effective eukaryotic expressing vector-pIRES1neo which contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer and the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV). HFCL cells were transfected with the recombinant vector pIRES1neo/hGM-CSF by liposome-mediated gene transfer method. Integration of hGM-CSF in the genome, transcription of its mRNA and expression of its protein in the transfected HFCL cells were assayed by Southern blot, Northern blot, ELISA and hGM-CSF dependent cell line TF-1.
Results:
hGM-CSF cDNA was integrated into HFCL genome successfully, hGM-CSF mRNA was transcripted and hGM-CSF protein was expressed of (56.9 +/- 0.7) ng/10(6) cells by ELISA and (6.56 +/- 0.16) x 10(3) U/10(6) cells per day by TF-1 cell assay in the supernatant.
Conclusion:
The recombinant vector is proved to be stably expressed in HFCL cells and the biological activity of hGM-CSF was detectable in the supernatant of the transfected cells.