Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene.
Leukemic cell lines such as HL-60, U937, and KG-1 provide an excellent model for studying human myeloid differentiation. These cells can be induced to differentiate from their immature state to form cells resembling more morphologically and functionally mature monocytes, macrophages, and granulocytes. During differentiation, expression of gene products such as myeloperoxidase and the integrin cell surface antigen CD11b is decreased or increased, respectively. Thus, these cell lines constitute an excellent model system in which to study the regulation of such differentially expressed genes. However, these myeloid cell lines are refractory to transfection by calcium phosphate or diethylaminoethyl (DEAE) dextran. Here we have optimized the transient transfection of myeloid cell lines using electroporation and the firefly luciferase reporter gene driven by viral promoters. The luciferase assay is extremely sensitive; transcription that is not detectable by Northern blot or run-on assays can be measured with this system. The system can be used in combination with the inducing agent 12-o-tetradecanoylphorbol-13-acetate (TPA), thus allowing analysis of developmentally regulated genes in these cells. Preliminary results suggest that this system can be applied to study the promoter for the myeloid specific gene, CD11b.
Pahl
H L
HL
Hematology/Oncology Division, Beth Israel Hospital, Boston, Massachusetts.
Burn
T C
TC
Tenen
D G
DG
D001358:Avian Sarcoma Viruses / Q000235:genetics; D002454:Cell Differentiation; D003587:Cytomegalovirus / Q000235:genetics; D004560:Electricity; D005786:Gene Expression Regulation; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate