Introduction

Inducible regulation of transgene expression in mammalian cells is a valuable tool in the study of gene function. Control of both the timing and level of protein expression not only minimizes potentially detrimental consequences of high, long-term overexpression on cell signaling and viability (1), but also increases the likelihood of observing physiologically relevant cellular effects.

The most commonly used inducible protein expression systems are those regulated by tetracycline (Tet) and its derivatives. Various Tet-regulated expression systems have been developed, including repression systems using the Tet-repressor (TetR) protein to block target gene transcription in the absence of Tet (2), or transactivation systems using TetR-mammalian transcription factor fusion derivatives to either switch transcription of a target gene on or off in response to Tet (Tet-On and Tet-Off systems) (3,4). While possessing many advantages over other current systems, basal leakiness still remains the main problem reported with these Tet controlled systems (5,6). The high basal expression levels in Tet systems are most probably due to the site of chromosomal integration, which has been described to be an important factor in tight regulation of the Tet promoter (7). False promoters or cryptic initiation signals may also contribute to a leaky expression of the Tet system under noninduced conditions (8).

Various approaches have been developed to tighten the control of gene expression of the Tet-regulated systems. Most involve the reduction of gene dosage either by low–copy number episomal vector systems (9,10) or by single-copy chromosomal integration through the use of retroviral vectors (11,12,13). Others have tried to decrease basal activity of the promoter by means of point mutations. However, results were generally disappointing as these modified systems do not sustain a high level of gene expression (14,15). Other strategies, which have met with some success, have involved the use of more efficient repressors and the use of combinations of different repressors to reduce basal levels while maintaining good induction ratios (16,17,18,19). Here, we report an alternative novel, simple, and broadly applicable method to overcome basal leakiness of inducible expression systems. We show, using Tet-inducible expression of two distinct sphingosine kinases as model systems, that incorporation of AU-rich mRNA destabilizing elements (AREs) in the 3′ untranslated region (UTR) of inducible constructs results in a significant decrease in the leakiness of the Tet-inducible expression system while maintaining high levels of inducibility.

Materials and Methods

Materials

Dulbecco's modified Eagle's medium (DMEM), HEPES buffer solution, penicillin, and streptomycin were purchased from CSL Biosciences (Parkville, Australia) and tetracycline-free fetal bovine serum (FBS) was obtained from BD Biosciences (Palo Alto, CA, USA). Protease inhibitors (Complete) were purchased from Roche Diagnostics GmbH (Mannheim, Germany); doxycycline from Sigma Aldrich (St. Louis, MO, USA); Flp-In T-Rex HEK293 cells, pOG44 recombinase, and Benchmark pre-stained protein standards from Invitrogen (Carlsbad, CA, USA); nitrocellulose membranes from Schleicher and Schuell (Keene, NH, USA); D-erythro-Sphingosine from Biomol Research Laboratories, Inc. (Plymouth, PA, USA); and [γ³²P]ATP and [α³²P]dATP from Perkin Elmer (Melbourne, Australia).

Construction of Expression Plasmids

The human sphingosine kinase 1 and 2 (SK1 and SK2) cDNAs containing C-terminal FLAG epitope tags (20,21) were cloned into pcDNA5/FRT/TO (Invitrogen) by digestion with KpnI and NotI. Subsequently, AREs (underlined) were incorporated into the 3′ UTRs of these plasmids using the oligonucleotides 5′-GGCCGCATTTATTTATTTATTTATTTAGGTACCTGCAGTTTATTTATTTATTTATTTAAGCTTC-3′ and 5′-TCGAGAAGCTTAAATAAATAAATAAATAAACTGCAGGTACCTAAATAAATAAATAAATAAATGC-3′ (Geneworks, Adelaide, Australia). These oligonucleotides were heated at 95°C for 5 min, and annealed by cooling to room temperature. These AREs were then ligated into pcDNA5/FRT/TO, pcDNA5/FRT/TO-SK1, and pcDNA5/ FRT/TO-SK2 following digestion with NotI and XhoI, leaving the multiple cloning site largely intact. Constructs were sequenced to verify incorporation of the desired AREs.

Cell Culture and Generation of Stably Transfected Inducible HEK293 Cell Lines

Flp-In T-Rex HEK293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 0.2% (w/v) sodium bicarbonate, 1.2 mg/mL penicillin, and 1.6 mg/mL streptomycin. The cells were co-transfected with the pOG44 vector encoding the Flp recombinase, and pcDNA5/FRT/ TO-, pcDNA5/FRT/TO-AU-, pcDNA5/ FRT/TO-SK1-, pcDNA5/FRT/TO-SK1-AU-, pcDNA5/FRT/TO-SK2-, or pcDNA5/FRT/TO-SK2-AU-inducible mammalian expression constructs in 9:1 ratio using the Lipofectamine 2000 reagent (Invitrogen) as described by the manufacturer. Two days after transfection the cells were passaged and after the cells had attached, the growth medium was replaced with a selective medium containing 150 µg/mL Hygromycin B and 15 µg/mL Blasticidin (Invitrogen). The selective medium was changed every 3 to 4 days until the desired number of cells was grown. Experiments were performed with pools of Hygromycin B–resistant cells, which by the nature of the Flp-In system are isogenic (22).