Human VNO cDNA libraries

Abstract

This invention relates to DNA libraries, in particular a human VNO cDNA library is described. Pheromone receptor cDNA once isolated is transfected into competent cells. The transfected cell lines provide a scaleable source of homogeneous material to develop efficient, automated high throughput screening assays for new vomeropherins, and thereby reduce the ongoing need for human volunteers in the preclinical phases of drug discovery. Identification and characterization of the human VNO receptor(s) will facilitate the development and commercialization of vomeropherins with improved specificity, and enhanced therapeutic efficacy in the treatment of the target diseases.

1 . A human VNO cDNA library constructed from female tissue ATCC # PTA-1213.  
     
     
         2 . A human CDNA library of female VNO tissue characterized by the presence of cDNAs coding for neuron-specific enolase, protein gene product 9.5 and synaptophysin, and the absence of cDNA coding for olfactory marker protein, wherein cDNA inserts range from about 300 base pairs to about 3000 base pairs in length.  
     
     
         3 . A human cDNA library of female VNO tissue characterized by the presence of cDNAs encoding Ga proteins G i1 , G i2 , G i3  and G olf .  
     
     
         4 . A human cDNA library of female VNO tissue characterized by the presence of cDNAs encoding adenylyl cyclase types 2, 3 and 7.  
     
     
         5 . cDNA corresponding to PP32 comprising SEQ ID Nos. 1 and 2.  
     
     
         6 . cDNA corresponding to PP33 comprising SEQ ID No. 3.  
     
     
         7 . cDNA corresponding to PP35 comprising SEQ ID No. 4 and 5.  
     
     
         8 . cDNA corresponding to PP38 comprising SEQ ID No.6.  
     
     
         9 . cDNA corresponding to PP40 comprising SEQ ID No. 16.  
     
     
         10 . cDNA corresponding to PP41 comprising SEQ ID No. 17.  
     
     
         11 . A method of identifying cDNA inserts encoding pheromone receptors comprising: 
 (a) generating a cDNA library which contains clones carrying cDNA inserts from human female VNO;    (b) hybridzing nucleic acid molecules of the clones from the cDNA libraries generated in step (a) with probes prepared from the group consisting of G-protein-coupled receptors, rodent VNO receptors and human pseudogene sequences;    (c) selecting clones which hybridized with the probes; and    (d) isolating clones which carry the hybridized inserts, thereby identifying the inserts encoding pheromone receptors.    
     
     
         12 . The method of  claim 11 , wherein the probes are cDNA probes.  
     
     
         13 . The method of  claim 11 , wherein the probes are cRNA probes.  
     
     
         14 . The method of  claim 11 , wherein the probes are genomic DNA probes.  
     
     
         15 . The method of  claim 11 , wherein the probes are PCR amplicons.  
     
     
         16 . The cDNA inserts identified by the method of claims  11 ,  12 ,  13 ,  14  or  15 .  
     
     
         17 . A method for identifying DNA inserts encoding pheromone receptors comprising: 
 (a) generating DNA libraries which contain clones carrying inserts from a sample containing human female vomeronasal organ tissue;    (b) contacting clones from the DNA libraries generated in step (a) with a nucleic acid molecule selected from the group consisting of G protein-coupled receptors, rodent VNO receptors and human pseudogene sequences, in appropriate conditions permitting hybridization of the cloned DNA and the nucleic acid molecule;    (c) selecting clones which hybridized with the nucleic acid molecule; and    (d) isolating the clones which carry the hybridized inserts, thereby identifying the inserts encoding the pheromone receptors.    
     
     
         18 . A method to identify DNA inserts encoding pheromone receptors comprising: 
 (a) Generating DNA libraries which contain clones with inserts from a sample which contains at least one human female vomeronasal organ;    (b) Contacting the clones from the DNA libraries generated in step (a) with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized inserts by polymerase chain reaction;    (c) Selecting the amplified inserts; and    (d) Isolating the amplified inserts, thereby identifying the inserts encoding the pheromone receptors.    
     
     
         19 . A method of  claim 18 , wherein the sample contains only human female vomeronasal organ cells.  
     
     
         20 . A method of  claim 17 , wherein the libraries are cDNA libraries.  
     
     
         21 . A method of  claim 18 , wherein the libraries are cDNA libraries.  
     
     
         22 . A method of  claim 17 , wherein the libraries are genomic DNA libraries.  
     
     
         23 . A method of  claim 18 , wherein the libraries are genomic DNA libraries.  
     
     
         24 . DNA inserts identified by the method of  claim 17 ,  18 ,  19 ,  20 ,  21 ,  22  or  23 .  
     
     
         25 . A method to isolate DNA molecules encoding pheromone receptors comprising: 
 (a) Contacting a biological sample known to contain nucleic acids with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized molecules by polymerase chain reaction; and    (b) Isolating the amplified molecules, thereby identifying the DNA molecules encoding the pheromone receptors.    
     
     
         26 . A method of  claim 25 , wherein the nucleic acid contained in the sample is DNA.  
     
     
         27 . A method of  claim 26 , wherein the nucleic acid contained in the sample is genomic DNA.  
     
     
         28 . The nucleic acid molecules isolated by the method of  claim 25 ,  26  or  27 .  
     
     
         29 . A method to isolate DNA molecules encoding pheromone receptors comprising: 
 (a) Contacting a biological sample known to contain nucleic acids with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized molecules by Reverse Transcriptase (RT)-polymerase chain reaction; and    (b) Isolating the amplified molecules, thereby identifying the DNA molecules encoding the pheromone receptors.    
     
     
         30 . A method of  claim 29 , wherein the nucleic acid contained in the sample is RNA.