tet-responsive seap promoter  (ARIAD Inc)

Journal: BMC Biotechnology

Article Title: A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

doi: 10.1186/1472-6750-4-9

Figure Lengend Snippet: A. Schematic of system . DBD (ZFHD) and AD (p65) fused components are co-expressed either as a single unit from one CMV promoter using an IRES element, or are separated to two different plasmids (DBD from pAr-DBD and AD from pAr-AD). In the presence of dimerizer, these elements activate expression of a ZFHD-responsive reporter (hGH or SEAP) and the tTA protein. In the absence of tetracycline, tTA activates expression of gene 2 (SEAP or GFP).

Article Snippet: In terms of explaining the moderate reduction in dynamic range versus the ARIAD system, we believe that this is most simply attributable to the degree of basal transcriptional activity obtained from the Tet-Responsive SEAP promoter, as similar total values of SEAP activity were obtained from DDC and SEAP systems, while reporter manifested detectable SEAP activity even in the absence of any additional components (Figure , column D).

Techniques: Expressing

Journal: BMC Biotechnology

Article Title: A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

doi: 10.1186/1472-6750-4-9

Figure Lengend Snippet: Optimization of induction signal . A mixture of 3 plasmids (pC 4 N 2 -R H S/ZF3, pZBS-Tet-OFF, and pBI-EGFP-SEAP) was used for cell transfection; the total amount of plasmid DNA was constant at 0.5 μg per test point. As graphically represented, at each apex of the triangle shown, only one of the test plasmids is present (100% of mix), while opposite the matrix this plasmid is absent (0% of mix), with the two other plasmids present in equal proportions. White circles indicate ratios of plasmids evaluated for degree of induction of SEAP activity. Degree of induction reflects difference between growth without dimerizer and with tetracycline (OFF), versus with dimerizer and without tetracycline (ON). Based on the ON:OFF ratios obtained, the software program "Statistica" modeled the optimal response surface, shown as a contour plot on the triangle. Faded edges of the triangle represent limitations of the model, because it is meaningless whenever one of the components is absent. The optimal ratio for plasmid transfection with the DDC system was 1.5:1.5:1 (pC 4 N 2 -R H S/ZF3:pZBS-Tet-OFF:pBI-EGFP-SEAP). This contrasted with a ratio of 3:1 (activator proteins:reporter) used for the original ARIAD system.

Article Snippet: In terms of explaining the moderate reduction in dynamic range versus the ARIAD system, we believe that this is most simply attributable to the degree of basal transcriptional activity obtained from the Tet-Responsive SEAP promoter, as similar total values of SEAP activity were obtained from DDC and SEAP systems, while reporter manifested detectable SEAP activity even in the absence of any additional components (Figure , column D).

Techniques: Transfection, Plasmid Preparation, Activity Assay, Software

Journal: BMC Biotechnology

Article Title: A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

doi: 10.1186/1472-6750-4-9

Figure Lengend Snippet: Demonstration of dual drug control . In Cos7 and HeLa cells, optimized ratios for the parental Ariad dimerizer System ( A ), tTA TET-OFF System (pRetro-OFF) ( B ), the merged Dual Drug Control (DDC) system ( C ), and the Tet-SEAP reporter in isolation ( D ) are evaluated for activity in the presence or absence of dimerizer and/or tetracycline. Values shown reflect luminometer readings of SEAP reporters. Blue boxes reflect values obtained in Cos7 cells; yellow boxes, in HeLa cells. Standard deviation is indicated by error bars. Each experiment was repeated at least three times.

Article Snippet: In terms of explaining the moderate reduction in dynamic range versus the ARIAD system, we believe that this is most simply attributable to the degree of basal transcriptional activity obtained from the Tet-Responsive SEAP promoter, as similar total values of SEAP activity were obtained from DDC and SEAP systems, while reporter manifested detectable SEAP activity even in the absence of any additional components (Figure , column D).

Techniques: Control, Isolation, Activity Assay, Standard Deviation

Journal: BMC Biotechnology

Article Title: A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

doi: 10.1186/1472-6750-4-9

Figure Lengend Snippet: Inducing gene expression at two tiers . Target genes are modeled by the hGH gene (ZFHD-dependent transactivation, expression responsive to dimerizer only; scale is on the right Y-axis) and the SEAP gene (tTA-dependent transcription expression responsive to dimerizer AND tetracycline; scale is on the left Y-axis). All cells contain pC 4 N 2 -R H S/ZF3, pZ 12 I-hGH-2, pZBS-Tet-OFF, and pBI-EGFP-SEAP. See Results for details of manipulations A-D; briefly, A was grown in continuous dimerizer-/tetracycline+; B-D were initially in dimerizer+/tetracycline+. At 48 hours after transfection with plasmids, A and B were harvested, while C and D were grown for 48 more hours in dimerizer+/tetracycline+ (C) or dimerizer+/tetracycline- (D) medium before harvesting. Results of one typical experiment, with four parallel transfections for each data point, are shown. Each experiment was repeated at least three times.

Article Snippet: In terms of explaining the moderate reduction in dynamic range versus the ARIAD system, we believe that this is most simply attributable to the degree of basal transcriptional activity obtained from the Tet-Responsive SEAP promoter, as similar total values of SEAP activity were obtained from DDC and SEAP systems, while reporter manifested detectable SEAP activity even in the absence of any additional components (Figure , column D).

Techniques: Gene Expression, Expressing, Transfection

Journal: BMC Biotechnology

Article Title: A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

doi: 10.1186/1472-6750-4-9

Figure Lengend Snippet: IRES versus split expression of two-hybrid components. Units shown represent fold-induction, induced over uninduced conditions, of a ZFHD-dependent SEAP reporter, in three different cell lines.

Article Snippet: In terms of explaining the moderate reduction in dynamic range versus the ARIAD system, we believe that this is most simply attributable to the degree of basal transcriptional activity obtained from the Tet-Responsive SEAP promoter, as similar total values of SEAP activity were obtained from DDC and SEAP systems, while reporter manifested detectable SEAP activity even in the absence of any additional components (Figure , column D).

Techniques: Expressing