Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells.
Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1alpha (EF-1alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.
Gopalkrishnan
R V
RV
Department of Urology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
Christiansen
K A
KA
Goldstein
N I
NI
DePinho
R A
RA
Fisher
P B
PB
D000818:Animals; D002999:Clone Cells; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D020648:Peptide Elongation Factor 1 / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics; D014162:Transfection / Q000379:methods; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D020648:Peptide Elongation Factor 1; D008156:Luciferases; D013752:Tetracycline