Part:BBa_K2597003

CMV

BBa_K2597003 expresses CMV promoter, a constitutive promoter for use in mammalian cells. Based on the work of Team Ljubljana 2007 BBa_I712004, BBa_K2597003 is constructed as mutated CMV P2 by changing the second natural NF-kB binding site into high-affinity SELEX-selected artificial sequence GGGGATTCCC, leading to the improvement of transcriptional activity.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design

CMV is known as the strongest promoter in mammalian cells and utilized widely in mammalian expression systems. There are four NF-kB binding sites on CMV. We mutated the second natural NF-kB binding site into artificial sequence GGGGATTCCC in order to improve the transcriptional activity.

Fig.1. Schematic of wt and mut P2 CMV. Boxes represent the sequences and relative positions of kBs in CMV. P, sequence used to create mut CMV.

Evaluation of optimized promoter activity with EGFP

To evaluate the transcriptional activity of promoters in a visualized format, we used EGFP as a reporter gene. The pEGFP vectors cloned with wt CMV and optimized mut CMV P2 were used to transfect CHO and HepG2 cells. The results reveal that EGFP expression was strongly driven by the promoters in the two cells. (Figure 2)

Fig.2. Detection of promoter activity by using EGFP as reporter gene. The HepG2 and CHO cells were transfected with the pEGFP plasmids containing different promoters. Cells were photographed under the bright field (left) and the fluorescence (right) with a fluorescence microscopy at 200 magnification. WT, wt CMV; CON, control (cells transfected with no plasmid).

Evaluation of optimized promoters with multiple cells

To test if the optimized mut CMV P2 also had high transcriptional activity in various cells, we evaluated the transcriptional of wt CMV and mut CMV P2 in five different mammalian cells lines, including HepG2, HeLa, K562, CHO and 293T, by using the dual-luciferase reporter assay and the GLuc reporter assay.

The dual-luciferase reporter assay revealed mut CMV P2 always had stronger transcriptional activity than wt CMV in five detected cells while GLuc reporter assay indicated that mut CMV P2 consistently outperformed wt CMV, verifying the higher transcriptional of mut CMV in various cells.

Fig.3. Evaluation transcriptional activity of promoters with multiple cells. (A and B) Detection of transcriptional activity of promoters by using the Dual-Luciferase reporter assay (A) and GLuc reporter assay (B) at 24 h post transfection. RLA, relative luciferase activity; RC, reporter constructs; NC, negative control. Statistical significance: mut CMV versus wt CMV.

Methods

Cells were seeded at a density of 1×105 cells per well in 24 well plates and cultivated for 12 h. For the first experiment, Cells were transfected with the pEGFP vectors (0.8 mg/well) containing the interested promoters by using Lipofectamine 2000 for 5 h. Then the media were discarded and the cells were cultured with fresh medium for more time. Cells were imaged with fluorescence microscope IX51 equipped with a DP71 or a laser scanning confocal microscope at a magnification of 200×. Cells were trypsinized for measuring the mean fluorescence intensity (MFI) by using flow cytometry.

For the second experiment, cells were cotransfected with pGL4.10 (0.5 mg/well) containing the interested promoters or blank pGL4.10 (0.5 mg/well) (as negative control) and internal control pGL4.75 (0.05 mg/well) by using Lipofectamine 2000 for 5 h for the Dual-Luciderase reporter assay. The media were then discarded and the cells were cultured with fresh media for more time. Cells were collected for detecting Fluc by using the Dual-Luciferase Reporter Assay System (Promega). The optical density was read with SynergyHT. The promoter activity was assessed by the relative Fluc activity normalized against the Renilla luciferase activity. For GLuc reporter assay, cells were transfected with the pGluc (0.8 mg/well) containing the interested promoters or blank pGluc (as negative control) by using Lipofectamine 2000 for 5 h. Then the media were discarded and cells were cultured for more time with fresh medium. The culture media were collected for measuring the Gluc activity by using the reagents for detecting the GLuc activity. The total protein concentration of culture media were determined by the Bradford Protein Assay Kit. The optical density was read with SynergyHT. The promoter activity was assessed with the relative GLuc activity normalized against the total protein concentration.