Anticipated Results & Significance
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Transfected cells have now TLR receptor (4 or 5) and our construct under NF-kappaB promoter. We expect that after stimulation (with LPS or flagellin) cytoplasmic NF-kappaB become active, migrate into nucleus and induce expression of our construct. Accumulating of dominant negative protein should block downstream signalling pathway. Concentration of active transcription factor should decrease and no more product should be synthesized. The rate of transcription and translation should be detected with detection systems - decrease of luminescence, free NF-kapaB and::: (depends on detection system). In case that our construct is followed by PEST (degradation) sequence, it is expected that product should degrade. If stimulus is still present, NF-kappaB will become active. Our product will be synthesized again. | Transfected cells have now TLR receptor (4 or 5) and our construct under NF-kappaB promoter. We expect that after stimulation (with LPS or flagellin) cytoplasmic NF-kappaB become active, migrate into nucleus and induce expression of our construct. Accumulating of dominant negative protein should block downstream signalling pathway. Concentration of active transcription factor should decrease and no more product should be synthesized. The rate of transcription and translation should be detected with detection systems - decrease of luminescence, free NF-kapaB and::: (depends on detection system). In case that our construct is followed by PEST (degradation) sequence, it is expected that product should degrade. If stimulus is still present, NF-kappaB will become active. Our product will be synthesized again. | ||
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Revision as of 21:53, 19 October 2006
| Backround and Signalling Pathway | Proposal & Approach | Anticipated Results & Significance | Troubleshooting, References & Sponsors | Team members |
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Anticipated results and their significance for understanding the mechanism of immune response
Transfected cells have now TLR receptor (4 or 5) and our construct under NF-kappaB promoter. We expect that after stimulation (with LPS or flagellin) cytoplasmic NF-kappaB become active, migrate into nucleus and induce expression of our construct. Accumulating of dominant negative protein should block downstream signalling pathway. Concentration of active transcription factor should decrease and no more product should be synthesized. The rate of transcription and translation should be detected with detection systems - decrease of luminescence, free NF-kapaB and::: (depends on detection system). In case that our construct is followed by PEST (degradation) sequence, it is expected that product should degrade. If stimulus is still present, NF-kappaB will become active. Our product will be synthesized again.
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