Difference between revisions of "Part:BBa K415509"

Revision as of 21:21, 28 October 2010

pNR1NR2_SV40 Shear Stress Responsive Promoter

L4R1 MammoBlock Promoter Vector. Preliminary data suggests that this synthetic promoter lends transcriptional sensitivity to fluid shear stress in the HEK293FT cell line. The schematic for the construction of this part, based on Silberman et al., Angiogenesis, 2009, is shown below. The promoter consists of a concatenation of three different shear stress responsive motifs isolated from different genes in front of the SV40 minimal promoter.

Design of the NR1NR2_SV40 promoter

• PDGF/B: the original shear stress responsive element (SSRE) isolated from the platelet derived growth factor B promoter and has sequence GAGACC.
• Tissue Factor: Sp1 site isolated from the Tissue Factor promoter and has sequence GGGGCGGGGCG.
• MCP-1: the TRE site isolated from the promoter of teh MCP-1 gene. It has the sequence TGACTCC.
• SV40: Minimal promoter

Characterization

Figure 1. Shear stress response of NR1NR2_SV40_EGFP.

Transient tranfections of pMech_EGFP constructs were performed on HEK293FT cells seeded at 2 million cells/well in two identically seeded six-well plates. The constructs used were pNR1NR2_SV40_EGFP and CMV_EGFP. CMV_EGFP served as a control for transformation efficiency.

12 hours after transfection one of the two six well plates was designated as "shear stress" and placed on a shaker at 120 rpm inside the incubator. The other plate was designated "control" and not subjected to shaking. Fluorescent micrographs were obtained on a Zeiss microscope for each well at 100, 215, and 500 ms exposure times every 3 hours. Brightfield images were exposed for 6 ms.

Images were exported from the accompanying Axiovision software at identical histogram levels for each cell line. For image analysis, accurate cell count could not be obtained because HEK cells grow at high density. Instead area occupied by cells was used as an analogous measurement for fields of view with similar % confluence and measured with imageJ, an image analysis software provided by the NIH. Fluorescent micrographs were processed using the binary function in imageJ, followed by particle analysis to obtain the total area occupied by fluorescence. The ratio of the area of fluorescence and the area of cells were calculated (R_fl) for time points 2.5h, 8h, 15.5h, and 24.5h. The overall ratio R_o was calculated as R_fl(T)/R_fl(2.5), where T=8,15.5, or 24.5. In this process the image levels and cell confluence of each micrograph were carefully matched for comparison.

Results

(Figure 1) NR1NR2_SV40 experienced significant upregulation in transcriptional activity (1.96x) under low level shear stress, as compared to negative control, which experienced no shear stress.

Sequence and Features