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Cloning-free regulated monitoring of reporter and gene expression.

al-Haj L, Al-Ahmadi W, Al-Saif M, Demirkaya O, Khabar KS - BMC Mol. Biol. (2009)

Bottom Line: Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Biomolecular Research, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia. lalhaj@kfshrc.edu.sa

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.

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Related in: MedlinePlus

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 (log0.5) 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.
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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 (log0.5) 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.

Mentions: The cloning-free approach (Figure 3A) was easily applied to generate a PCR product expressing EGFP reporter under the control of metal responsive element (MRE)-mediated transcription. Minimal metal response elements [8] that belong to metallothioneins promoters, MT1F and MT1G (Table 1), were included in the Forward primer. The heavy metal, cadmium, was used to upregulate the transcription of the MRE-containing minimal promoter from the transfected EGFP reporter PCR product (Figure 3A). At 10 uM of cadmium, response from MT1F, which contains three MRE sites was stronger than that of MT1G, which has two MRE sites. However, at 20 uM, both MREs mediate similar responses.


Cloning-free regulated monitoring of reporter and gene expression.

al-Haj L, Al-Ahmadi W, Al-Saif M, Demirkaya O, Khabar KS - BMC Mol. Biol. (2009)

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 (log0.5) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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 (log0.5) 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.
Mentions: The cloning-free approach (Figure 3A) was easily applied to generate a PCR product expressing EGFP reporter under the control of metal responsive element (MRE)-mediated transcription. Minimal metal response elements [8] that belong to metallothioneins promoters, MT1F and MT1G (Table 1), were included in the Forward primer. The heavy metal, cadmium, was used to upregulate the transcription of the MRE-containing minimal promoter from the transfected EGFP reporter PCR product (Figure 3A). At 10 uM of cadmium, response from MT1F, which contains three MRE sites was stronger than that of MT1G, which has two MRE sites. However, at 20 uM, both MREs mediate similar responses.

Bottom Line: Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Biomolecular Research, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia. lalhaj@kfshrc.edu.sa

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.

Show MeSH
Related in: MedlinePlus