Cloning-free regulated monitoring of reporter and gene expression

Abstract

Background: The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations.

Results: In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes.

Conclusion: The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

Figure 1

Schematic representation of the cloning-free reporter construction and assay and use in inflammation model. (A) An expression plasmid harboring a reporter cDNA is used as a template for PCR. Forward primer sequence contains 3' end that targets a minimal promoter region and 5'end that accommodates cis-acting sites. The reverse universal primer targets a region downstream of the polyA site. Purified PCR products are transfected into mammalian cells to express the reporter.(B) HEK293 cells in 96-well plates were transfected with 75 ng of purified PCR products in which the forward primer contains two copies of NF-κB site or a mutant form (Table 1, SEQ 1 and 2). After 20 hr, the cells were treated with IL-1α (log_0.5) for additional 20 hrs. (B, insert). Dose-response relationships between IL-1 and NF-κB mediated GFP reporter activity (normalized to basal levels of mutant NF-κB reporter levels). (C) EGFP activity was measured after 4 hr of IL-1 treatment with or without normalization (insert) with non-responsive RFP-PCR product. All data above are Mean ± SEM (quadruplicate) of one of three experiments. **p < 0.01, *** < 0.001. (D) Total RNA was extracted from HEK293 cells that were transfected with NF-κB responsive or mutant NF-κB-bearing PCR products in the absence or presence of IL-α (10 ng/ml) for 4 or 6 hrs. RT-PCR was performed using primers specific to EGFP as described in Methods. PCR products were run on gel, and β-actin normalized signal intensities of ethidium bromide-stained products were quantitated using AlphaEase software. Data are from three experiments.(E) Dose-dependent curve of TNF-α action on reporter activity produced from NF-κB responsive linear PCR construct.

Figure 2

Dynamic response of the activity of reporter expressed from PCR product. (A) The reporter expression plasmid was used as a template for PCR in which the forward primers target either -53 or -95 sequences of the CMV promoter and includes two or four copies of NF-κB or a mutant NF-κB control (Table 1, SEQ. 1–3). (B) Fluorescence levels from EGFP PCR products that contain two, four, or mutant copies of NF-κB were quantitated as described above.(C) HEK293 cells were transfected with either NfκB-firefly luciferase expression vector or purified PCR products that were amplified from the luciferase vector and co-transfected with Renilla luciferase expression vector. Empty DNA represent either the vector or the PCR product without the inserted NF-κB sequence. The cells were treated with either IL-1α or TNF-α for 4 hrs as previously described. Luciferase activity levels were quantified by a luminometer and either absolute expression levels (C) or fold increase over control (no treatment) are shown (D). Data are Mean ± SEM. *p < 0.05, *** p < 0.001.

Figure 3

Generation of metal and IFN-responsive reporter PCR products and their performance. (A) HEK293 cells in 96-well plates were transfected with 75 ng of purified PCR products generated from reporter vector using the Forward primer that contains MRE sites (Table 1, SEQ. 4 and 5). After 20 hr, the cells were treated with 10 μM cadmium for 16 hrs. Data is Mean ± SEM (quadruplicate) – a representative experiment of two- of fold increase due to cadmium in GFP fluorescence levels that were normalized to background fluorescence from non-MRE PCR product. (B) Huh7 cells in 96-well plates were transfected with 75 ng of purified PCR products in which the Forward primer includes two putative ISRE sites (Table 1 SEQ 6). After approximately 20 hr, the cells were treated with IFN-α (log_0.5) for an additional 16 hrs. Right panel: An IFN-resistant HEK293 cells were transfected with the ISRE-containing EGFP PCR product (20 hr) and then treated with IFN-α (33 IU/ml) for 16 hr. Data are from one experiment (Mean ± SEM (n = 4). (C) Dose-response curve for IFN action on GFP reporter activity from the ISRE-containing PCR product (normalized to fluorescence levels from non-responsive reporter). *, p < 0.01, **p < 0.005 and ***p < 0.0001. (D) Total RNA was extracted from Huh7 cells that were transfected with IFN-responsive reporter PCR products in the presence or absence of IFN-α (100 U/ml) for 4 or 6 hrs. RT-PCR was performed using primers specific to EGFP as described in Methods. PCR products were run on gel, and β-actin normalized signal intensities of ethidium bromide-stained products (Mean ± SEM (n = 3) were quantitated using AlphaEase.

Figure 4

Cloning-free construction and evaluation of de novo minimal promoters. (A, B) The reporter expression plasmid was used as a template for PCR. Forward primer contains at 3' end, 18 bases that target 5'UTR (exon 1 of the CMV IE gene) upstream of reporter cDNA and 5' end sequence representing 48 bases derived from CMV (control), RPS2, RPL39 promoters or the minimal inducible IFNB promoter (Table 1, SEQ 7–9). The reverse universal primer targets a region downstream of the polyA site. (C) The resultant and purified PCR products were transfected onto HEK293 cells. After 48 hrs, the GFP fluorescence was quantitated as outlined in the Figure 1 legend.(D) TLR-3 stably transfected HEK293 cells (TRL3+) or control (TLR3 negative) HEK293 cells were transiently transfected with IFNB minimal promoter-harboring PCR products and were treated with or without 25 μg/ml of poly I. C. Fluorescence levels were quantitated after 20 hr as described above.

Figure 5

Cloning-free conversion of genes into inducible-repressible expression cassette. (A) Schematic representation of the cloning-free conversion of a gene product into tetracycline-inducible gene. Forward primer contains 3' end sequences that targets a minimal region in the vector template and 5'end that contain two or three copies of TetO sites (Table 1, SEQ. 10–12). Reporter-C' MODC denotes unstable EGFP version that was created by fusing EGFP with MODC C-terminus as described in Methods. (B) HeLa Tet-off cells in 96-well plates were transfected with 75 ng of PCR products generated from reporter vector (A) and subsequently treated with or without 0.1 ug/ml of doxycycline for 26 hr. As a control, EGFP PCR product which lacks TetO was used. Arrows denotes % reduction. (C) Purified PCR products amplified from expression vectors that contain either wild type EGFP or unstable EGFP were transfected onto HeLa Tet-off cells in the presence or absence of 0.1 ug of doxycycline for 26 hrs. Data in B and C are Mean ± SEM (quadruplicate) from a representative experiment of three. (D) Representative images of the effect of doxycycline on the TetO-containing reporter expression PCR products with or without normalization of RFP-expressing PCR product that lack TetO sites. Images represent single (I) or co-transfection (II) experiments. (E) HeLa Tet-off cells were transfected with purified PCR products that were amplified from TTP expression vector with TetO₂ forward primer (Table 1, SEQ. 11) and polyA reverse primer, in the absence or presence of 0.1 ug/ml of doxycycline. Western blotting using anti-body to TTP and GAPDH was performed. V denotes vector only.