Journal:

Article Title: Characteristics of the Adeno-Associated Virus Preintegration Site in Human Chromosome 19: Open Chromatin Conformation and Transcription-Competent Environment

doi:

Figure Lengend Snippet: Transcriptional activity of DHS-S1. (A) Structure and activity of constructs containing DHS-S1 cloned upstream of the CAT gene. The whole DHS-S1 was excised as an SmaI-SmaI fragment (nt 18 to 353 of AAVS1) from plasmid pRVK and cloned in both orientations (indicated by the direction of the arrows) upstream of the intron and the CAT gene into the SmaI site of pCAT 3-basic vector (Promega). Black boxes represent the SV40 promoter. The promoterless plasmid is the pCAT 3-basic vector (Promega), and the control plasmid containing the SV40 promoter is the pCAT 3-promoter vector (Promega). Regions FR1 (nt 1313 to 1616 of AAVS1) and FR3 (nt 2633 to 2927 of AAVS1) were amplified by PCR with plasmid pRVK as a substrate and oligonucleotides carrying SmaI sites at either end. The resulting fragments were digested with restriction enzyme SmaI and cloned into the SmaI site of the pCAT 3-basic vector. Region FR2 (nt 2077 to 2371 of AAVS1) was excised from plasmid pRVK as a BamHI-flanked fragment, filled in by treatment with Klenow enzyme, and inserted into the SmaI site of pCAT 3-basic vector. Ten micrograms of each plasmid was transfected as described previously by calcium phosphate precipitation into 293 and HeLa cells (1). At 15 h posttransfection, the medium was changed: after an additional 24 h, cells were collected and CAT activity in cell extracts was measured by using the Quan-T-CAT assay system (Amersham, Pharmacia). CAT activities were normalized against the luciferase activity derived from a cotransfected cytomegalovirus-luciferase plasmid (1). Results are expressed as milliunits of CAT enzyme present in 10 μg of cell extracts and as fold induction with respect to the promoterless pCAT 3-basic vector. The data are means ± standard deviations of two independent experiments in which two separate plasmid preparations were used. (B) Enhancer activity of DHS-S1 in 293 and HeLa cells. DHS-S1 was cloned in both orientations (represented by the direction of the arrows) into the pCAT 3-promoter vector either into the SmaI site upstream of the SV40 promoter or into the BamHI site downstream of the CAT gene. Similarly, FR1, FR2, and FR3 were cloned in one orientation either into the SmaI site or into the BamHI site of pCAT 3-vector. Transfections were performed, and CAT activities were measured and normalized as described in the legend to Fig. ​Fig.4A.4A. Results are expressed as milliunits of CAT enzyme in 10 μg of cell extracts; fold induction was calculated with respect to that of the pCAT 3-promoter vector. The data are means ± standard deviations of three independent experiments in which at least two different plasmid preparations were used.

Article Snippet: The whole DHS-S1 was excised as an Sma I- Sma I fragment (nt 18 to 353 of AAVS1) from plasmid pRVK and cloned in both orientations (indicated by the direction of the arrows) upstream of the intron and the CAT gene into the Sma I site of pCAT 3-basic vector (Promega).

Techniques: Activity Assay, Construct, Clone Assay, Plasmid Preparation, Amplification, Transfection, Luciferase, Derivative Assay

Journal:

Article Title: Characteristics of the Adeno-Associated Virus Preintegration Site in Human Chromosome 19: Open Chromatin Conformation and Transcription-Competent Environment

doi:

Figure Lengend Snippet: Enhancer activity of DHS-S1 is mainly located in the 181 to 353 region. (A) Graphic representation of the two DHS-S1 subregions, E1-S1 (nt 18 to 181) and E2-S1 (nt 181 to 353), whose transcriptional activity was tested in transient transfection assays. (B) Enhancer activity of E1-S1 and E2-S1 in 293 and HeLa cells. E1-S1 and E2-S1 were obtained as PCR fragments by using plasmid pRVK (see legend to Fig. ​Fig.1)1) as a template, and cloned in both orientations (indicated by the direction of the arrows) into plasmid pCAT 3-promoter either upstream of the SV40 promoter or downstream of the CAT gene (see the legend to Fig. ​Fig.4).4). Transfections, CAT assays, and normalization of the results were performed as described in the legend to Fig. ​Fig.4.4. Results are expressed as milliunits of CAT per 10 μg of cell extracts and as fold induction with respect to the pCAT 3-promoter vector. The data are means ± standard deviations of four independent experiments in which two different plasmid preparations were used.

Article Snippet: The whole DHS-S1 was excised as an Sma I- Sma I fragment (nt 18 to 353 of AAVS1) from plasmid pRVK and cloned in both orientations (indicated by the direction of the arrows) upstream of the intron and the CAT gene into the Sma I site of pCAT 3-basic vector (Promega).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Clone Assay

Journal:

Article Title: Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function

doi: 10.1016/j.bbagrm.2010.05.003

Figure Lengend Snippet: The constitutive and HNE-inducible activity of putative EpRE sequences in the 5′-flanking region of human GCLC gene. Oligos containing the corresponding EpRE were cloned into pGL-3 promoter vector and the reporters were transfected into HBE1 cells. After being treated with/without 15 μM HNE for 24h, cells were collected and the luciferase activity was measured. The luciferase activity was normalized with co-transfected β-Gal activity. N=3, * P< 0.05 compared with vehicle vector (v: pGL-3 promoter vector), # P< 0.05 compared with control treatment.

Article Snippet: Luciferase activity assay kit, pGL-3 bas ic vector, and pGL-3 promoter vector, were from Promega (Madison, WI).

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Control

Journal:

Article Title: Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function

doi: 10.1016/j.bbagrm.2010.05.003

Figure Lengend Snippet: Effect of EpRE location on EpRE activity. Reporters were constructed as described in the Experimental Procedurals. The reporters were transfected into HBE 1 cells and the luciferase activity was measured. The relative luciferase activity was calculated by using the vehicle vector (pGL-3 basic vector) without HNE exposure as the control. N=3, *, P< 0.05 compared with corresponding reporter without HNE exposure; #, P< 0.05 compared with GCLC-Luc without HNE exposure. GCLC-Luc, −3802/+85 GCLC-luc; m4 GCLC-Luc, −3802/+85 GCLC-Luc with EpRE 4 mutated; in EpRE 4/n1-Luc, EpRE 4/n6-Luc, EpRE 4 was relocated to the position of EpRE n1, and n6 of m4 GCLC-Luc, respectively; in EpRE n1/E4-Luc, EpRE n6/E4-Luc and EpRE 3/E4-Luc, EpRE 4 in GCLC-Luc was replaced with EpRE n1, n6, and EpRE 3, respectively.

Article Snippet: Luciferase activity assay kit, pGL-3 bas ic vector, and pGL-3 promoter vector, were from Promega (Madison, WI).

Techniques: Activity Assay, Construct, Transfection, Luciferase, Plasmid Preparation, Control

Journal:

Article Title: Upregulation of cytochromes P 450 2B in rat liver by orphenadrine

doi: 10.1038/sj.bjp.0705305

Figure Lengend Snippet: HepG2 cells were transfected with the cyp2b10 gene PBREM-luciferase reporter construct or cotransfected with the PBREM-luciferase reporter construct and a plasmid encoding the mCAR. In addition, all cells were cotransfected with a plasmid encoding β-galactosidase. Transfected cells were treated with androsten-3α-o1 (AND; 8 μM) to suppress endogenous CAR activation, ORPH (0.1 mM) or TCPOBOP (250 nM), or combinations of these reagents. Results are fold induction of reporter-gene activity in AND-treated cells after correction for pGL3 alone; data derived from three to four separate transfections. Different from AND-treated cells: **P<0.01, *P<0.05.

Article Snippet: The β -galactosidase plasmid, reagents for luciferase assay and pGL-3 basic vector were from Promega.

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Activation Assay, Activity Assay, Derivative Assay