Transfection Collection™ : PDE4D Transient Pack
The PDE4D Transient Pack is designed to provide the tools necessary for transiently transfecting and for screening inhibitors of PDE4D7 in cultured HEK293 cells.
The assay is based on transfecting cells with the CRE luciferase reporter. CRE reporter contains the firefly luciferase gene under the control of cAMP response element (CRE). Elevation of intracellular cAMP activates CRE binding protein (CREB) to bind CRE and induce the expression of luciferase. Forskolin is commonly used to raise the intracellular level of cAMP in cell physiology studies. When cells transiently transfected with CRE reporter are activated by forskolin, the intracellular level of cAMP is upregulated, which induces the expression of CRE luciferase reporter. However, when cells are co-transfected with PDE4D7 expression vector and CRE reporter, the level of forskolin-induced cAMP is reduced, resulting in lower expression level of luciferase. When cells are treated with PDE4D inhibitor to inhibit PDE4D7 activity, cAMP level is restored, resulting in higher luciferase activity.
The PDE4D Transient Pack contains transfection-ready CRE luciferase reporter. This reporter contains transfection-ready vectors for PDE4D7 expression and a CRE vector containing firefly luciferase as a cAMP pathway-responsive reporter and constitutively expressing Renilla luciferase as a transfection control. It also includes the TWO-Step Luciferase detection reagents to detect both luciferase activities and specialized medium for growing and assaying HEK293 cells.
The key to the PDE4D Cell-Based Activity Reporter Detection Valuepack is the CRE luciferase reporter vector, which is a cAMP/PKA Cell Signaling Pathway-responsive reporter. This reporter contains the firefly luciferase gene under the control of multimerized cAMP response elements (CRE) located upstream of a minimal promoter. The vector is premixed with constitutively-expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency. The pack also includes the PDE4D7 expression vector, and the adenylate cyclase activator, forskolin. Additionally, the pack includes cell culture medium (BPS Medium 1) that has been optimized for use with HEK293 and HeLa cells*. BPS Medium 1 includes MEM medium, 10% fetal bovine serum, 1% non-essential amino acids, sodium pyruvate, and 1% Pen/Strep. Finally, the pack provides the TWO-Step Luciferase (Firefly & Renilla) Assay System. These luciferase reagents provide highly sensitive, stable detection of firefly luciferase activity and Renilla luciferase activity. The TWO-step luciferase reagents can be used directly in cells in growth medium, and can be detected with any luminometer; automated injectors are not required.
*Note: the kit may be used with other cell lines than HEK293 or HeLa, but an alternate cell culture medium may be required for optimal cell growth
Purchase of this cell line is for research purposes only; commercial use requires a separate license. View the full terms and conditions.
| Component | Amount | Storage |
| Reporter (Component A) CRE luciferase reporter vector* + constitutively expressing Renilla luciferase vector* | 500 µl (60 ng DNA/ µl) | -20°C |
| PDE4D7 expression vector (Component B) | 250 µl (40 ng DNA/µl) | -20°C |
| Firefly Luciferase Reagent Buffer | 10 ml | -20°C |
| Firefly Luciferase Reagent Substrate (100x) | 100 µl | -20°C Protect from light |
| Renilla Luciferase Reagent Buffer | 10 ml | Room Temp. |
| Renilla Luciferase Reagent Substrate (100x) | 100 µl | -20°C Protect from light |
| BPS Medium 1 | 100 ml | +4°C |
*These vectors are designed for transient transfection. They are NOT suitable for transformation and amplification in bacteria.
Phosphodiesterases (PDEs) play an important role in the dynamic regulation of cAMP and cGMP signaling. PDE4D has 3',5'-cyclic-AMP phosphodiesterase activity and degrades cAMP. Inhibition of PDE4D activity by its inhibitors leads to an elevated intracellular level of cAMP. The PDE4D gene encodes at least 9 different isoforms, and has been linked to stroke, asthma, arrhythmia, and cardiac myopathy, making it an important therapeutic target.
2. Fan Chung, K. (2006) Phosphodiesterase inhibitors in airways disease. Eur. J. Pharmacol. 533(1-3):110-117
3. Malik, R. et al. (2008) Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors. Appl. Microbiol. Biotechnol. 77 (5): 1167-1173
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