A novel tetracycline-dependent expression vector with low basal expression and potent
regulatory properties in various mammalian cell lines
A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines
Anke
Hoffmann
,
Martin
Villalba
1
,
Laurent
Journot
1
and
Dietmar
Spengler*
Max Planck Institute of Psychiatry, Molecular Neurobiology, Kraepelinstrasse 2-10, D-80804 Munich, Germany and
1
CNRS UPR 9023, Centre CNRS-INSERM de Pharmacologie-Endocrinologie (CCIPE), 141 rue de la cardonille, F-34094 Montpellier Cedex 05, France
Received November 18, 1996;
Revised and Accepted January 17, 1997
ABSTRACT
The tetracycline-dependent expression system has gained increasing popularity for the
expression of any gene of interest. Careful choice of the expression vector has been suggested to exploit the full potential of this system. A novel tetracycline-sensitive expression vector based on a modified mouse mammary tumor virus promoter achieved
considerably improved regulatory properties in a series of cell lines tested
under transient and stable conditions. Therefore, the applicability of the
tetracycline-dependent expression system can be largely enhanced by careful adaptation of the expression vector to the host cell line.
Briefly, the tetracycline-dependent expression system relies on the tetracycline-responsive transcriptional regulatory element of
Escherichia coli
(tetO) and a bacterial
tet
-repressor protein. This repressor protein is fused to the C-terminal domain of the protein VP16 from herpes simplex virus which
serves as a potent transactivator to stimulate transcription from minimal
promoter sequences that are positioned downstream of the
tet-
operator sequence. Transcription from such a promoter was almost completely
suppressed when tetracycline was present in the culture medium, whereas
expression is readily achieved by removal of the repressor tetracycline (
1
,
1
). Beside the ability to tightly control transcription of heterologous genes in
transiently or stably transfected cells, the levels of basal expression are
critical to study gene products altering cell viability or cell proliferation
or to be studied in a temporal fashion due to desensitization or adaptation
under continued expression (
4
). We used the experience we gained upon development of a cAMP-responsive reporter system with lowest possible levels of basal expression
and high induction ratios to circumvent limitations from high basal levels of
expression and poor regulatory properties we observed for the original CMV
based minimal promoter in various cell lines tested. A modified mouse mammary
tumor virus promoter ([Delta]MTV) in which the steroid-responsive region was replaced by multiple copies of a cAMP-responsive element (CRE) proved to confer exquisite
sensitivity to cAMP in several cell lines (
4
and references therein). We decided, therefore, to transfer the tetO sequences
into this promoter and to compare regulatory properties of the new construct p[Delta]MtetO-Luc with those of the CMV-based pUHC13-3 plasmid (
1
, hereafter called pCMVtetO-Luc) in cell lines from various origin. To create p[Delta]MtetO, the regulatory region of pCMVtetO was excised by
Hin
dIII and partial
Eco
RI digest and inserted into pBluescript SK- digested by
Eco
RI and
Hin
dIII to give pBSK-CMVtetO. A fragment of 157 bp encoding the tetO sequence was released by
Sma
I and inserted into the plasmid [Delta]MTVLUC (
4
) linearized at +256 bp by
Hin
dIII and blunted with T4-DNA polymerase to give the construct p[Delta]MtetO-Luc.
As shown in Table
1
, p[Delta]MtetO-Luc-transfected cells displayed 12-100-fold lower basal luciferase levels depending on
the cell line and time after transfection. This lower basal luciferase activity
resulted in higher induction ratios in all cell lines and at all time points.
Though the maximal luciferase activities in the induced condition with p[Delta]MtetO-Luc were lower than the ones obtained in pUHC13-3-transfected cells, the induction ratios were, but for
the HeLa cell line, preserved at 24 and 48 h for p[Delta]MtetO-Luc in contrast to pCMVtetO-Luc, which, dependent on the cell type, either decreased (LLC-PK1, SaOs-2, C6) or increased (HeLa, SK-N-MC, CV1).
REFERENCES
1 Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89, 5547-5551.MEDLINE Abstract
2 Gossen, M. and Bujard, H. (1993) Trends Biochem. Sci.18,471-475.
3 Gossen, M. and Bujard ,H. (1995) BioTechniques19, 213-216.MEDLINE Abstract
4 Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeeburg, P. H. and Journot, L. (1993) Nature365, 170-175.MEDLINE Abstract
*To whom correspondence should be addressed at present address: CNRS UPR 9023,
Centre CNRS-INSERM de Pharmacologie-Endocrinologie (CCIPE), 141 rue de la cardonille, F-34094 Montpellier Cedex 05, France. Tel: +33 467 14 29 32;
Fax: +33 467 54 24 32; Email: spengler@ccipe.montp.inserm.fr
