Difference between revisions of "Part:BBa K415510"

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==Characterization==
 
==Characterization==
  
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The response of K415510 to mechanical stimulation via fluid shear stress  
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The response of K415510 to mechanical stimulation via fluid shear stress:
  
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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: pCOFS_EGFP, pPAI-2_EGFP, pSRE/CRE2_SV40_EGFP, pNR1/2_SV40_EGFP, and CMV_EGFP. Of these 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.
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[[Image:SRE_Characterization.png]]
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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 pSRE/CRE2_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.
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'''Results'''
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SRE/CRE2 exhibited a significant increase in activity (2.4x) compared to the negative control, with no applied shear stress.

Revision as of 21:09, 27 October 2010

CRE2 is a hybrid promoter, constructed by placing elements from the mechanosensitive FosB promoter in front of an SV40 constitutive promoter. It's been shown that ERK-dependent activation of CREB binding to a CRE/AP-1 like element (designated “CRE2”) in the FosB promoter is one of the main effectors of mechanosensitivity; SRE, or Shear Responsive Element, was first characterized as a mechanosensitive element in the cfos promoter.

Characterization

The response of K415510 to mechanical stimulation via fluid shear stress:

SRE Characterization.png

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 pSRE/CRE2_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

SRE/CRE2 exhibited a significant increase in activity (2.4x) compared to the negative control, with no applied shear stress.