Nucleic Acids Research, Vol 27, Issue 24 4775-4782, Copyright © 1999 by Oxford University Press
RV Gopalkrishnan, KA Christiansen, NI Goldstein, RA DePinho and PB Fisher
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.
ARTICLES
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
Department of Urology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
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