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Combinatorial marking of cells and cell structures with reconstituted fluorescent proteins

總結: Lead Inventors: Martin Chalfie, PhDProblem or Unmet Need:Fluorescent proteins, first developed as biological markers by Dr. Chalfie, have become a key reagent in the molecular biology toolbox. Green Fluorescent Protein (GFP) and its derivatives are used as reporters because they readily form active fusion proteins, can be safely expressed in cells and are easily visualized by standard microscopic techniques. Current applications of fluorescent proteins, especially high throughput screens used in drug discovery, are limited in spatial and temporal resolution by the number of flourophores. This invention adds finer grain control to the expression of fluorescent molecules; dramatically improving assays.This invention details the use of a "split" GFP, where the original GFP molecule is expressed as two separate polypeptides. When brought into close proximity, the polypeptides fold together, reconstituting the original protein and its fluorescent properties. The key to this invention is the low rate of spontaneous reconstitution; the polypeptides will not fluoresce without a molecular "zipper" to associate them together. This "zipper" is modular since binding domain from protein-protein, protein-small molecule or protein-nucleic acid interactions can be used to reconstitute the fluorescent protein. Also, the polypeptides can be introduced into animal models using standard transgenic techniques, providing a direct path for in-vivo target validation.

技術優勢: GFP is a mature technology with an established base of molecular and imaging techniques Dual promoter expression allows unique tissue or temporal specificity in assay design Modularity of assays, the technology is amenable to using combinatorial libraries of proteins, peptides or small molecules

技術應用: Gene expression in development and disease -- by linking split GFP expression to two promoters of interest, assays can be developed which pinpoint expression in tissues. Drug Discovery -- identification of small-molecule to protein interactions by fluorescence, where interaction partners are tethered to split GFP Drug Efficacy -- by limiting expression of split GFP to certain tissues/cell types under control of a specific promoter, the effect of drug candidates can be measured with high resolution Regulatory circuit discovery -- using a library approach, uncharacterized promoters can be described by coordinate expression with known promoters. Recovery of cells identified in any screen is possible with fluorescence assisted cell sorting Fusion to immunoglobulin can simplify screening of hybridomas

詳細技術說明: This invention details the use of a "split" GFP, where the original GFP molecule is expressed as two separate polypeptides. When brought into close proximity, the polypeptides fold together, reconstituting the original protein and its fluorescent pro...

*Inquiry: Jullian Jones Columbia Technology Ventures Tel: (212) 854-8444 Email: TechTransfer@columbia.edu

*Publications: Zhang S, Ma S and Chalfie M. Combinatorial Marking of Cells and Organelles with Fluorescent Proteins. (2004) Cell. 119:137-144.

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