Difference between revisions of "Part:BBa K415512"

Latest revision as of 22:35, 28 October 2010

pCFOS L4R1 MammoBlock Entry Vector

L4R1 MammoBlock Promoter Vector. The c-fos proto-oncogene promoter in mammalian cells is suspected of containing regulatory mechanosensory elements in the region [-356,0], where 0 designates the transcriptional start site. Previously, Peake and Haj (FEBS, 2003) found cyclic strain to induce actin stress fiber formation and induction of mRNA for c-fos within an hour of application.

The c-fos promoter sequence in the region [-500,189] was isolated via genomic PCR and inserted into the standard L4R1 MammoBlock Promoter Vector.

Characterization

Figure 1. Shear stress response of CFOS_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 pCFOS_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, the c-fos promoter experienced little difference in transcriptional activation due to fluid shear stress within the time frame of the experiment. The c-fos promoter was not expected to respond to fluid shear stress but primarily to cyclic, nonuniform mechanical strain, as characterized by previous literature. We believe that further experimentation in cyclic strain for this promoter will generate significant results.

Our results established that, as hypothesized, the c-fos promoter does not respond to fluid shear stress. It is capable of being trasncriptionally active at an appreciable level in the HEK293FT cell line.

Sequence and Features

Relevant Literature

Peake and Haj. "Preliminary characterisation of mechanoresponsive regions of the c-fos promoter in bone cells." FEBS Letters, 2003.