Gene gun transfection of human glioma and melanoma cell lines with genes encoding human IL-12 and GM-CSF.
We used particle-mediated gene transfer by a custom-built gene gun to transfect two well-established human glioma (D54MG and U251) and melanoma (SK mel 28 and Ed 141) cell lines, as well as two glioma lines locally established from primary patient tumors (Ed 147 and Ed 149). Using beta-galactosidase as a reporter gene, D54MG, U251, Ed 141 and SK mel 28 showed an average transfection efficiency of 15-40%, whereas Ed 147 and Ed 149 had mean transfection efficiencies of 3% and 5% respectively. Twenty-four hours after transfection with the gene encoding human interleukin-12 (IL-12), ELISA was performed on cell supernatants (mean of n = 12 for each cell line). IL-12 expression was extremely variable between the different cell lines, ranging from 52 to 1,151 pg/10(6) cells/24 h. Results were very similar when cells were exposed to 20,000 rads of gamma irradiation 2 h after transfection. When the cell lines were transfected with human granulocyte-macrophage colony-stimulating factor, 24 h levels were: 13.0 (Ed 147), 17.8 (Ed 149), 18.6 (Ed 141), 27.4 (D54MG) and 27.7 ng/10(6) cells/24h (U251). SK mel 28 produced 88.1 ng/10(6) cells/24 h. We conclude that the gene gun can efficiently transfect a variety of immortalized, well-established and locally-established glioma and melanoma cell lines. High dose gamma irradiation does not adversely affect the expression of the foreign gene (IL-12) at 24 h. Significantly, transfected cell lines show different levels of expression depending on the particular gene/plasmid introduced. Therefore, each cell line has to be assessed individually for the level of expression of each introduced gene.
Gainer
A L
AL
Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada. againer@ualberta.ca
Young ATL
Parney
I F
IF
Petruk
K C
KC
Elliott
J F
JF
D002461:Cell Line, Transformed; D015870:Gene Expression; D017930:Genes, Reporter; D005910:Glioma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics*; D008545:Melanoma; D014162:Transfection / Q000295:instrumentation* / Q000379:methods; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D018664:Interleukin-12; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D001616:beta-Galactosidase