Part:BBa_K415511
pPAI2 L4R1 MammoBlock Entry Vector
L4R1 MammoBlock Promoter Vector. The PAI2 (Plasminogen Activator Inhibitor 2) promoter is suspected of containing regulatory mechanosensory elements, specifically cyclic, nonuniform strain. The sequence from the human PAI2 [-2000,249], where 0 designates the transcriptional start site, was isolated via genomic PCR.
Characterization
Response of K415511 to mechanical stress via low level nonuniform fluid shear stress.
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 pPAI2_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 and Conclusion
As seen in Figure 1, PAI2 experienced significant upregulation in transcriptional activity early in the experiment at 8hr (2.5x) under low level shear stress, as compared to negative control, which experienced no shear stress. However, overall expression of EGFP was very low throughout the experiment.
PAI2 was not expected to respond to fluid shear stress but primarily to cyclic, nonuniform mechanical strain. We believe that while the early activation of this promoter may be significant, more experiments must be performed for strain in order to confidently classify this part as mechanosensitive.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal XbaI site found at 145
Illegal SpeI site found at 55 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal SpeI site found at 55 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal BglII site found at 1082
Illegal BglII site found at 1402 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal XbaI site found at 145
Illegal SpeI site found at 55 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal XbaI site found at 145
Illegal SpeI site found at 55 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1313
Illegal BsaI site found at 1578
Illegal BsaI.rc site found at 674
Illegal SapI.rc site found at 1016
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