# US 5,705,349 A · Methods for preparing polynucleotides encoding orphan receptor ligands > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,705,349 A - Document ID: US5705349A - Status: Granted - Kind: A - Application number: 08/490,803 - Filed: 1995-06-15 - Granted: 1998-01-06 - Earliest claimed: 1994-02-14 - Assignees: ZymoGenetics, Inc. - Inventors: Richard D. Holly; Steven K. Burkhead - Web page: https://patentgrep.com/patent/US5705349A ## CPC - G01N 33/502: {for testing non-proliferative effects} - C07K 14/47: from mammals - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - C07K 14/715: for cytokines; for lymphokines; for interferons - C12N 5/0642: {Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells} - G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)} - G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics} - A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells} - C12N 2501/145: Thrombopoietin [TPO] - C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Methods for obtaining cells that produce a ligand for an orphan receptor and methods for preparing polynucleotide molecules that encode ligands for orphan receptors are disclosed. The methods utilize growth factor-dependent parent cells that are transfected with a DNA construct encoding an orphan receptor. The transfected cells are exposed to mutagenizing conditions, and the mutagenized cells are cultured under conditions in which cell survival is dependent upon autocrine growth factor production. Progeny cells are recovered and screened to identify those that produce a ligand for the orphan receptor. Polynucleotide molecules encoding the ligand can be prepared from the identified cells. ## Claims ### Claim 1 A method for preparing polynucleotide molecules wherein at least one of said molecules encodes a ligand for an orphan growth factor receptor comprising: (a) providing parent cells, wherein growth of said cells is dependent upon an exogenous growth factor; (b) transfecting said parent cells with a DNA construct encoding an orphan growth factor receptor to produce transfected cells expressing said orphan growth factor receptor encoded by said DNA construct; (c) exposing said transfected cells to mutagenizing conditions to produce mutagenized cells; (d) culturing said mutagenized cells under conditions in which cell survival is dependent upon autocrine growth factor production; (e) recovering progeny cells that survive said culturing step; (f) screening said progeny cells to identify cells that produce a ligand for said orphan growth factor receptor; and (g) preparing polynucleotide molecules from said identified cells wherein at least one of said molecules encodes said ligand. ### Claim 2 (depends on 1) A method according to claim 1 wherein said polynucleotide is cDNA. ### Claim 3 (depends on 1) A method according to claim 1 wherein said polynucleotide is mRNA. ### Claim 4 (depends on 1) A method according to claim 1 wherein said polynucleotide is genomic DNA. ### Claim 5 (depends on 1) A method according to claim 1 wherein said parent cells do not express detectable levels of said orphan growth factor receptor. ### Claim 6 (depends on 1) A method according to claim 1 wherein, prior to said culturing step, said mutagenized cells are cultured in the presence of said exogenous growth factor. ### Claim 7 (depends on 1) A method according to claim 1 wherein said screening step comprises assaying media conditioned by said progeny cells for growth-promoting activity on said transfected cells. ### Claim 8 (depends on 7) A method according to claim 7 wherein said assay is carried out in the presence of an antibody to a known growth factor. ### Claim 9 (depends on 7) A method according to claim 7 wherein said assay is carried out in the presence of a soluble form of said orphan growth factor receptor. ### Claim 10 (depends on 1) A method according to claim 1 wherein said exposing step comprises exposure to a chemical mutagen. ### Claim 11 (depends on 1) A method according to claim 1 wherein said parent cells are myeloid or lymphoid progenitor cells. ### Claim 12 (depends on 1) A method according to claim 1 wherein said molecules of step (g) are an expression library and said method further comprises: (h) transfecting said expression library into suitable host cells; (i) screening conditioned media of said host cells to identify cells producing said ligand; and (j) isolating polynucleotide molecules encoding said ligand from the cells identified in step (i).