Functional expression of, and assay for, functional cellular receptors in vivo

Abstract

Methods and materials for expressing and assaying functional neuronal receptors in neuronal cells, including methods for detecting particular odorant ligand specificity for particular odorant receptors and methods of using such. For example, methods and materials are provided for assaying for functional odor receptors in intact nasal epithelium of mammals such as rats and mice and for using such.

What is claimed is:  
     
         1 . A method for screening for cellular receptors having a particular receptor ligand specificity comprising the steps of: 
 expressing at least one recombinantly produced cellular receptor in a cell located amongst intact tissue;    contacting said recombinantly produced cellular receptor expressing cell with at least one candidate ligand; and    assaying for functional interaction between said recombinantly produced cellular receptor and said candidate ligand.    
     
     
         2 . The method of  claim 1 , wherein said recombinantly produced cellular receptor is expressed in said cell in vivo.  
     
     
         3 . The method of  claim 1 , wherein said recombinantly produced cellular receptor is expressed in said cell in situ.  
     
     
         4 . The method of  claim 1 , wherein said cell is a differentiated cell.  
     
     
         5 . The method of  claim 1 , wherein said cell is an olfactory sensory neuron.  
     
     
         6 . The method of  claim 1 , wherein said cell is in a mammal's nasal epithelium.  
     
     
         7 . The method of  claim 1 , wherein the cellular receptor is selected from the group consisting of the superfamily of G protein coupled receptors.  
     
     
         8 . The method of  claim 1 , wherein the cellular receptor is a neuronal receptor.  
     
     
         9 . The method of  claim 1 , wherein the cellular receptor is an odorant receptor.  
     
     
         10 . The method of  claim 9 , wherein the odorant receptor is odorant receptor I7.  
     
     
         11 . The method of  claim 1 , wherein the contact of said recombinantly produced cellular receptor expressing cell with at least one candidate ligand is via airborne administration of said candidate ligand.  
     
     
         12 . The method of  claim 1 , wherein said candidate ligand is selected from the group consisting of cellular receptor agonist molecules and cellular receptor antagonist molecules.  
     
     
         13 . The method of  claim 1 , wherein the candidate ligand is an odorant molecule.  
     
     
         14 . The method of  claim 1 , wherein the candidate ligand is selected from the group consisting of the ligands identified in Table 1.  
     
     
         15 . The method of  claim 1 , wherein the recombinantly produced cellular receptor is odorant receptor I7 and the candidate ligand is selected from the group consisting of heptyl aldehyde, octyl aldehyde, nonyl aldehyde, and decyl aldehyde.  
     
     
         16 . The method of  claim 1 , wherein the recombinantly produced cellular receptor is produced from a recombinant construct comprising a cloned cellular receptor and an adenovirus.  
     
     
         17 . The method of  claim 16 , wherein the adenovirus is human adenovirus type 5 and the cellular receptor is odorant receptor I7.  
     
     
         18 . The method of  claim 1 , wherein said assay for functional interaction comprises measuring the change in membrane potential of one or more of said cells in response to said candidate ligand.  
     
     
         19 . The method of  claim 1 , wherein the assay for functional interaction between said recombinantly produced cellular receptor and said candidate ligand comprises the step of: 
 measuring the electrical activity of one or more of said cells containing said recombinantly produced cellular receptor in response to said candidate ligand.    
     
     
         20 . The method of  claim 19 , wherein said electrical activity is measured by means of extracellular recording, intracellular recording, recording en passant, whole-cell recording using patch clamp, and single channel recording.  
     
     
         21 . The method of  claim 20 , wherein said cell is a single cell.  
     
     
         22 . The method of  claim 1 , further wherein: 
 said intact tissue containing said cell is isolated in vitro prior to contact with said candidate ligand.    
     
     
         23 . The method of  claim 1 , further wherein: 
 at least one recombinantly produced marker is expressed with the expression of said recombinantly produced cellular receptor in said cell, said marker thereby identifying cells containing said recombinant receptor.    
     
     
         24 . The method of  claim 23 , wherein said marker is selected from the group consisting of fluorescent markers and histochemical markers.  
     
     
         25 . A composition of matter comprising: 
 a receptor-ligand complex of recombinantly produced odorant receptor I7 and a ligand selected from the group consisting of straight chain aldehydes having from 7 to 10 carbons in length.    
     
     
         26 . The composition of  claim 25 , wherein the ligand is octyl aldehyde.  
     
     
         27 . The composition of  claim 25 , further comprising a neuronal cell in which said recombinantly produced odor receptor I7 has been produced and expressed.  
     
     
         28 . The composition of  claim 27 , wherein the neuronal cell is a nasal epithelial cell.  
     
     
         29 . A method for screening for neuronal receptors having a particular receptor ligand specificity comprising the steps of: 
 expressing at least one recombinantly produced neuronal receptor in a neuronal cell located amongst intact tissue;    isolating intact tissue expressing said recombinantly produced neuronal receptor;    contacting said recombinantly produced neuronal receptor expressing cell with at least one candidate ligand; and    assaying for functional interaction between said recombinantly produced neuronal receptor and said candidate ligand.    
     
     
         30 . The method of  claim 29 , wherein said neuronal receptor is an odorant receptor, said neuronal cell is an olfactory cell, and said ligand is an odorant ligand.  
     
     
         31 . A method for screening for ligands having a particular cellular receptor specificity comprising the steps of: 
 expressing at least one recombinantly produced cellular receptor in a cell located amongst intact tissue;    contacting said recombinantly produced cellular receptor expressing cell with at least one candidate ligand; and    assaying for functional interaction between said recombinantly produced cellular receptor and said candidate ligand.