Method for extracting quantitative information relating to an influence on a cellular response
US8058008B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Feb 5, 2002 |
| Grant date | Nov 15, 2011 |
| Priority date | — |
| Expiry date | May 6, 2025 |
Classification
- Technology area (CPC G)Physics
- CPC primaryG01N2333/9121
- WIPO fieldMeasurement
- WIPO sectorInstruments
Abstract
Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signalling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of the influence. Measurement of redistribution is performed by recording of light intensity, fluorescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a fluorescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method …
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