Epididymal lipocalin gene and uses thereof

Abstract

Isolated nucleic acids comprising a lipocalin gene promoter region, isolated nucleic acids comprising a human lipocalin gene, isolated nucleic acids encoding a lipocalin polypeptide, isolated lipocalin polypeptides, and uses thereof. The disclosed lipocalin nucleic acids and polypeptides can be used to generate a mouse model of male infertility, for drug discovery screens, and for therapeutic treatment of fertility-related conditions.

What is claimed is:  
     
         1 . An isolated promoter region, comprising an about 5.3 kb fragment (Accession No. AF082221) of mouse genomic clone 10983 (Genomesystem Inc., St. Louis, Ky.) between the EcoRV and SalI restriction sites, or functional portion thereof.  
     
     
         2 . An isolated promoter region of  claim 1 , the functional portion comprising a TATA box and at least one cis-acting regulatory sequence selected from the group consisting of a Sp1-binding site, an AP-1 binding site, a retinoic acid receptor binding site, an androgen receptor binding site, a C-Ets binding site, a SRY binding site, an AP-4 binding site, a C/EBP binding site, and combinations thereof.  
     
     
         3 . The isolated promoter region of  claim 1 , comprising: 
 (a) the nucleotide sequence of SEQ ID NO:1; or    (b) a nucleic acid molecule substantially identical to SEQ ID NO:1.    
     
     
         4 . The isolated promoter region of  claim 1 , comprising a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:1.  
     
     
         5 . An isolated promoter region, or functional portion thereof, comprising an about 5150 base pair region immediately upstream of the human hEP17 transcription start site.  
     
     
         6 . The isolated promoter region of  claim 5 , the functional portion comprising a TATA box and at least one cis-acting regulatory sequence selected from the group consisting of a Sp-1 binding site, an AP-1 binding site, a cAMP response element binding protein (CREB) binding site, a SRY-related HMG-box gene 5 (Sox5) binding site, a Sex-determining region Y gene product (SRY) binding site, a c-Ets binding site, a GATA binding site, an Octamer transcription factor 1 (Oct-1) binding site, and combinations thereof.  
     
     
         7 . The isolated promoter region of  claim 5 , comprising: 
 (a) the nucleotide sequence of SEQ ID NO:5; or    (b) a nucleic acid molecule substantially identical to SEQ ID NO:5.    
     
     
         8 . The isolated promoter region of  claim 5 , comprising a 20 base pair nucleotide sequence identical to a contiguous 20 base pair nucleotide portion of SEQ ID NO:5.  
     
     
         9 . A chimeric gene comprising the isolated promoter region of  claim 1  or  5  operably linked to a heterologous nucleotide sequence.  
     
     
         10 . A vector comprising the chimeric gene of  claim 9 .  
     
     
         11 . A host cell comprising the chimeric gene of  claim 9 .  
     
     
         12 . The host cell of  claim 11 , wherein the cell is selected from the group consisting of a bacterial cell, a hamster cell, a mouse cell, and a human cell.  
     
     
         13 . A mouse comprising a mouse cell of  claim 12 .  
     
     
         14 . The mouse of  claim 13 , wherein expression of the chimeric gene confers male infertility in an otherwise fertile mouse.  
     
     
         15 . A method for identifying a substance that regulates EP17 expression, the method comprising: 
 (a) establishing a gene expression system comprising the chimeric gene of  claim 12 , wherein the heterologous nucleotide sequence is a reporter gene, and components required for gene transcription and translation, whereby the reporter gene is expressed, and a level of reporter gene expression is assayable;    (b) assaying a baseline level of reporter gene expression using the gene expression system of (a) in the absence of a candidate substance;    (c) exposing the gene expression system of (a) to a plurality of candidate substances;    (d) assaying a level of reporter gene expression using the gene expression system of (a) in the presence of a candidate substance of (c); and    (e) selecting a candidate substance whose presence results in an altered level of reporter gene expression when compared to the baseline level.    
     
     
         16 . The method of  claim 15 , wherein the substance is a protein, a chemical compound, or a peptide.  
     
     
         17 . A method for identifying a substance that regulates EP17 expression, the method comprising: 
 (a) creating a transgenic mouse bearing a chimeric transgene comprising the promoter region of  claim 1  or  5  operably linked to a reporter gene, wherein the reporter gene is expressed, and wherein a level of reporter gene expression is assayable;    (b) assaying a baseline level of reporter gene expression in the transgenic mouse in the absence of a candidate substance;    (c) administering the candidate substance to the transgenic mouse;    (d) assaying a level of reporter gene expression in the transgenic mouse following administration of the candidate substance to the mouse; and    (e) selecting a substance wherein the level of reporter gene expression in the transgenic mouse is altered following administration of the substance compared to the baseline level.    
     
     
         18 . A method for producing an epididymal cell line, the method comprising: 
 (a) creating a transgenic animal bearing a chimeric transgene gene comprising the promoter region of  claim 1  operably linked to a selectable marker gene that permits cell growth in the presence of a selective agent;    (b) procuring epididymal cells from the transgenic animal of (a); and    (c) reproducing the cells in vitro in the presence of the selective agent.    
     
     
         19 . A method for mutagenizing an EP17 locus in a vertebrate animal, the method comprising: 
 (a) constructing a targeting vector having the isolated promoter region of  claim 1 , a marker gene, and an isolated 3′ flanking region of an EP17 gene, wherein the marker gene is positioned between the promoter region and the 3′ flanking region;    (b) linearizing the targeting vector by digestion with a restriction endonuclease, wherein the promoter region, marker gene, and 3′ flanking region are undigested;    (c) introducing the linearized vector into embryonic stem cells;    (d) detecting the marker gene in the embryonic stem cells;    (e) selecting embryonic stem cells having the marker gene; and    (f) generating a transgenic vertebrate animal derived from the selected embryonic stem cells, wherein the EP17 locus of the animal is altered as a result of a homologous recombination event mediated by the targeting vector.    
     
     
         20 . An isolated EP17 polypeptide, or functional portion thereof, comprising: 
 (a) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:3;    (b) a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3;    (c) a polypeptide having the amino acid sequence of SEQ ID NO:4;    (d) a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or    (e) a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NO:4    
     
     
         21 . An isolated nucleic acid molecule encoding a human EP17 polypeptide.  
     
     
         22 . The isolated nucleic acid molecule of  claim 21 , comprising: 
 (a) the nucleotide sequence of SEQ ID NO:3; or    (b) a nucleic acid molecule substantially identical to SEQ ID NO:3.    
     
     
         23 . The isolated nucleic acid molecule of  claim 21 , comprising a 20 nucleotide sequence that is identical to a contiguous 20 nucleotide sequence of SEQ ID NO:3.  
     
     
         24 . A chimeric gene, comprising the nucleic acid molecule of  claim 21  operably linked to a heterologous promoter.  
     
     
         25 . A vector comprising the chimeric gene of  claim 24 .  
     
     
         26 . A host cell comprising the chimeric gene of  claim 24 .  
     
     
         27 . The host cell of  claim 26 , wherein the cell is selected from the group consisting of a bacterial cell, a hamster cell, a mouse cell, and a human cell.  
     
     
         28 . A method of detecting a nucleic acid molecule that encodes an EP17 polypeptide, the method comprising: 
 (a) procuring a biological sample having nucleic acid material;    (b) hybridizing the nucleic acid molecule of SEQ ID NO:1, 2, 3, or 5 under stringent hybridization conditions to the biological sample of (a), thereby forming a duplex structure between the nucleic acid of SEQ ID NO:1, 2, 3, or 5 and a nucleic acid within the biological sample; and    (c) detecting the duplex structure of (b), whereby an EP17 nucleic acid molecule is detected.    
     
     
         29 . An antibody that specifically recognizes an EP17 polypeptide encoded by the nucleotide sequence of SEQ ID NO:3; a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3; a polypeptide having the amino acid sequence of SEQ ID NO:4; a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NOs:4.  
     
     
         30 . A method for producing an antibody that specifically recognizes an EP17 polypeptide, the method comprising: 
 (a) recombinantly or synthetically producing an EP17 polypeptide of  claim 20 , or portion thereof;    (b) formulating the polypeptide of (a) whereby it is an effective immunogen;    (c) administering to an animal the formulation of (b) to generate an immune response in the animal comprising production of antibodies, wherein antibodies are present in the blood serum of the animal; and    (d) collecting the blood serum from the animal of (c) comprising antibodies that specifically recognize an EP17 polypeptide.    
     
     
         31 . A method for detecting a level of EP17 polypeptide, the method comprising 
 (a) obtaining a biological sample having peptidic material;    (b) detecting an EP17 polypeptide in the biological sample of (a) by immunochemical reaction with the antibody of  claim 29 , whereby an amount of EP17 polypeptide in a sample is determined.    
     
     
         32 . A method for identifying a substance that modulates EP17 function, the method comprising: 
 (a) isolating an EP17 polypeptide encoded by the nucleotide sequence of SEQ ID NO:3; a polypeptide encoded by a nucleic acid molecule that is substantially identical to SEQ ID NO:3; a polypeptide having the amino acid sequence of SEQ ID NO:4; a polypeptide that is a biological equivalent of the polypeptide of SEQ ID NO:4; or a polypeptide which is immunologically cross-reactive with an antibody that shows specific binding with a polypeptide of SEQ ID NO:4;    (b) exposing the isolated EP17 polypeptide to a plurality of substances;    (c) assaying binding of a substance to the isolated EP17 polypeptide; and    (d) selecting a substance that demonstrates specific binding to the isolated EP17 polypeptide.    
     
     
         33 . A method for modulating EP17 function in a subject, the method comprising: 
 (a) preparing a pharmaceutical composition, comprising a substance identified according to the method of  claim 15 ,  17 ,  30 , or  32 , and a carrier;    (b) administering an effective dose of the pharmaceutical composition to a subject, whereby EP17 activity is altered in the subject.    
     
     
         34 . A method for modulating EP17 function in a subject, the method comprising: 
 (a) preparing a gene therapy vector having a nucleotide sequence encoding an EP17 polypeptide or a nucleotide sequence encoding a nucleic acid molecule, peptide, or protein that interacts with an EP17 nucleic acid or polypeptide; and    (b) administering the gene therapy vector to a subject, whereby the function of EP17 in the subject is modulated.    
     
     
         35 . The method of  claim 34  further comprising the EP17 promoter region of  claim 1  or  5 .  
     
     
         36 . A method for diminishing the fertile capacity of a subject, the method comprising: 
 (a) identifying a chemical compound, peptide, or antibody that interacts with the polypeptide of SEQ ID NO:4 or 6;    (b) preparing a pharmaceutical composition comprising the chemical compound, peptide, or antibody of (a) and a carrier; and    (c) administering an effective dose of the pharmaceutical composition to a subject, whereby the fertile capacity of the subject is diminished.    
     
     
         37 . A method for promoting fertility in a subject, the method comprising: 
 (a) identifying a chemical compound or peptide that interacts with the polypeptide of SEQ ID NO:4 or 6;    (b) preparing a pharmaceutical composition comprising the chemical compound or peptide of (a) and a carrier; and    (c) administering the pharmaceutical composition to a subject, whereby the fertility of the subject is improved.