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Identification of ligands by selective amplification of cells transfected with receptors

  • US 5,955,281 A
  • Filed: 11/07/1997
  • Issued: 09/21/1999
  • Est. Priority Date: 07/13/1993
  • Status: Expired due to Term
First Claim
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1. A method for detecting a mutant form of a receptor or mutant form of a signal transducing protein associated with said receptor, which mutant form mediates cell amplification in the absence of an agonist of said receptor and which activation of non-mutant form of said receptor or non-mutant form of said signal transducing protein mediates cell amplification in the presence of, but not the absence of, an effective concentration of said agonist, comprising(a) providing a library of DNAs expected to comprise at least one DNA sequence coding for the mutant form of said receptor or mutant form of a signal transducing protein;

  • (b) transfecting the DNAs of step (a) into cells, said transfected cells also comprising a transfected marker of cell amplification;

    (c) incubating a cell culture which comprises the transfected cells of step (b) and which also comprises non-transfected cells which do not comprise said transfected marker (i) in the absence of an agonist of said receptor or (ii) in the presence of a lower concentration of said agonist than the effective concentration required for the activation of the non-mutant form of said receptor or a non-mutated form of said signal transducing protein for a period of time sufficient to permit cell amplification of said transfected cells; and

    (d) determining the level of cell amplification of said transfected cells by measuring the level of marker in cell culture, increased levels of marker indicating the presence of a mutant form of said receptor or signal transducing protein in said cell culture, which mutant form mediates cell amplification in the absence of an agonist of the receptor or in the presence of a lower concentration of said agonist than the effective concentration required for the activation of the non-mutant form of said receptor or a non-mutated form of said signal transducing protein.

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