[Expression of lipofect AMINE mediated human GM-CSF eukaryotic expressing vector in HFCL cells].
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.


CONCLUSIONS
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.
Ma
L
L
Department of Hematology & Oncology, The First Clinical College, Norman Bethune University of Medical Sciences, Changchun 130021, China.
Wang
G
G
Li
L
L
Feng
K
K
Bai
L
L
Pei
X
X
D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008055:Lipids; D008081:Liposomes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D012333:RNA, Messenger; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA