Adjusting transgene expression levels in lymphocytes with a set of inducible promoters.
BACKGROUND
Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells.


METHODS
The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells.


RESULTS
The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels.


CONCLUSIONS
The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.
Danke
Christina
C
Department Biology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany.
Grünz
Xandra
X
Wittmann
Jürgen
J
Schmidt
Andreas
A
Agha-Mohammadi
Siamak
S
Kutsch
Olaf
O
Jäck
Hans-Martin
HM
Hillen
Wolfgang
W
Berens
Christian
C
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002469:Cell Separation; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D053650:Interleukin-3 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D051379:Mice; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000502:physiology*; D019076:Transgenes
D053650:Interleukin-3 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3