Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Abstract

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.

Figure 1

Schematic map of pRTS-1. The vector is described in detail in Results. SA and SD denote splice donor and acceptor sites. The rabbit β-globin intron and polyadenylation site is placed behind the luciferase gene (luc). The bicistronic expression cassette driving expression of rtTA^s-M2 and tTS^KRAB, separated by an internal ribosomal entry site (IRES), is placed behind the chicken β-actin intron and transcribed from a promoter/enhancer consisting of the mouse heavy chain intron enhancer (Eμ) and the chicken β-actin promoter (CAGp). P_tetbi-1 denotes the bidirectional tetracycline-regulated promoter, oriP the EBV episomal origin of replication carrying the family of repeats (FR) and the dyad symmetry element (DS). EBNA1, the EBV gene EBNA1; bla, β-lactamase; SVp, the SV40 early promoter; ori, the bacterial origin of replication derived from pMB1; and hyg, the hygromycin phosphotransferase gene.

Figure 2

Reduction of background by the expression of tTS^KRAB. (A) BJAB cells (10⁷) were transfected with 10 μg pRT-1 and pRTS-1 DNA by electroporation. Cells were left untreated or treated with 1 μg/ml Dox immediately after electroporation and luciferase activity measured in cell extracts after 48 h. The relative light units of untreated cells are given above the columns. (B and C) Three BJAB cell lines were transfected with pRT-1 (B) or pRTS-1 (C), expanded in the absence of Dox, and analysed ∼2 months after transfection. Cells were treated with Dox or left untreated and were analysed for luciferase and eGFP expression 48 h after Dox addition. The luciferase activities of uninduced cells and the factor of inducibility are presented in the left panels. The differences in the factor of inducibility of pRT-1- and pRTS-1-transfected cells are due to the background activity in the absence of Dox. Note that the pattern of eGFP induction is not uniform in the different BJAB cell lines, even within one cell line. This is most probably due to the fact that the cell lines arising in individual wells of 96-well plates are not of clonal origin.

Figure 3

Simultaneous equivalent gene expression from both sides of the bidirectional promoter visualized at the single cell level. (A) Staining control of untransfected Raji cells. In (B) and (D), the luciferase gene was replaced by a bicistronic expression cassette encoding activation-induced deaminase and a truncated NGF receptor separated by an internal ribosomal binding site (IRES). In (C), the eGFP gene was replaced by a truncated NGF receptor gene and the luciferase gene was deleted by inserting a short stuffer inserted bridging the two SfiI sites. NGF receptor and eGFP expression was monitored by FACS staining in the absence (B) and presence of 1 μg/ml Dox (C and D). The double-negative cells represent untransfected Raji cells.

Figure 4

BL30-B95.8 cells transfected with pRTS-1-M1 (M1 gene of influenza virus) elicit an antigen-specific CD4⁺ T cell response only in the presence and not in the absence of Dox. Transfected BL30-B95.8 cells were co-cultured with the M1-specific CD4⁺ T cell clone E5. T cell stimulation was monitored by measuring GM-CSF release into the medium.

Figure 5

TGR-1 cells were transfected with 6 μg pRTS-1 DNA and expanded in 200 μg/ml hygromycin B for 4 weeks either in the presence (A) or in the absence of Dox (B and C). Cells expanded in the absence of Dox were treated with 1 μg/ml Dox for 24 h (B), or left untreated (C). Cells were visualized by light microscopy (upper panels of A, B and C) and fluorescence microscopy (lower panels). In (D–F), TGR-1 cells were transfected with 6 μg of either the vector expressing luciferase (pRTS-1), or, instead of luciferase, the gene encoding the nucleolar protein Bop1 wt, or its N-terminal deletion mutant Bop1Δ. Transfected cells were expanded under hygromycin B selection (200 μg/ml) for 2 weeks and eGFP expression determined by flow cytometry 24 h after the addition of Dox (D and E). (F) Equal numbers of cells transfected with luciferase, Bop1, and Bop1Δ were plated, Dox added and cell numbers determined after 6 days. The data are presented as percentage of control cells expressing luciferase. (G) Episomal replication of the plasmids was visualized by gel electrophoresis in 1% agarose after lysis of the cells in the gel (22). To evaluate the copy number, different number of Raji cells were included as well as TGR-1 cells to which different amounts of pRTS-1 had been added. Lanes (a)–(e) contain TGR-1 cells transfected with pRTS-1 (a), and vector expressing the nucleolar proteins or mutants WDR12 (b), WDR12 ΔNle (c), Bop1 (d) and Bop1Δ (e).

Figure 6

Graded increase of eGFP expression of pRTS-1 transfected TGR-1 cells with increasing Dox concentration. TGR-1 cells transfected with pRTS-1 were plated in six-well plates and different concentrations of Dox added (A). The numbers depicted in the histograms represent ng/ml Dox. Mean fluorescence of eGFP expression was plotted versus the Dox concentration in a double logarithmic plot (B). Note that, although the peak of eGFP expressing cells is very broad at intermediate Dox concentrations (25–100 ng/ml), induction does not follow an ‘all or none’ pattern.

Figure 7

Inducibility of eGFP expression is impaired when pRTS-1-transfected BJAB cells are cultivated in the absence of Dox over 5 months. Panels A and C represent two different pRTS-1-transfected BJAB cell lines analysed for eGFP expression in the absence and presence of Dox after 2 months in culture. Panels B and D show the pattern of eGFP expression in the same BJAB-transfected cell lines after 5 months in culture.