Gene identification method

Abstract

A method for the identification of genes that are essential for the maintenance of specific cell phenotypes is disclosed. The method includes the initial step of identifying a cell type with a phenotype of interest. Gene inactivation is performed on an aliquot of cells of the cell type of interest. Selection is then performed to an aliquot of the cell culture to which gene inactivation has been applied. Cells which continue to maintain the phenotype following gene inactivation have not had the gene of interest inactivated whereas cells in which genes necessary for maintaining the phenotype have been inactivated have been lost. Utilizing subtraction analysis between treated and untreated aliquots the gene in the cells which has been inactivated that affects the phenotype of interest is identified. Genes that are identified by the method are also disclosed as well as antibodies directed against the gene product of the identified genes. Further a customized kit for the practice of the gene identification method is also disclosed.

What is claimed is:  
     
         1 . A method for the identification of genes that are essential for the maintenance of a specific cell phenotype comprising the steps of: 
 a) identifying a cell type with a phenotype of interest;    b) inactivating genes in the cell of interest with a gene inactivation means on an aliquot of a culture of the cell type;    c) applying selection means to an aliquot of the cell culture of step (b) and reserving an aliquot of unselected cells;    d) isolating the selected cells of step (c);    e) isolating the vectors from the selected cells and from the control unselected aliquot of cells;    f) isolating the gene inactivating elements from the vectors; and    g) utilizing differential analysis means to identify the genes inactivated in the cells in step (c) that affect the phenotype of interest.    
     
     
         2 . The method as set forth in  claim 1  wherein the phenotype of interest can be selected from the group consisting of phenotype relating to growth, phenotype relating to release of factors, phenotype relating to expression of factors and phenotype relating to cell function.  
     
     
         3 . The method as set forth in  claim 1  wherein the selection means can be selected from the group consisting of ability of cells to survive under specific culture conditions, ability of cells to express a specific measurable factors, detectable changes in cell structure, and differential gene expression.  
     
     
         4 . The method as set forth in  claim 1  wherein differential analysis means are selected from the group of methods consisting of differential display, representational differential analysis (RDA), suppressive subtraction hybridization (SSH), serial analysis of gene expression (SAGE), gene expression microarrays, nucleic acid chip technology, and direct sequencing.  
     
     
         5 . A method for the identification of genes that are essential for the maintenance of specific cell phenotypes including the steps of: 
 a) identifying a cell type with a phenotype of interest;    b) preparing an expression cDNA library from cells expressing the phenotype;    c) transfecting a cell culture of the cell type with expression vectors incorporating the expression cDNA library;    d) applying selection means to an aliquot of the transfected cell culture in step (c) and reserving an untreated aliquot;    e) isolating cells which continue to maintain the phenotype and isolating the expression vector, or the DNA insert found in the expression vector, from the cells maintaining the phenotype;    f) isolating the expression vector, or the DNA insert found in the expression vector from the reserved untreated aliquot of the cells; and    g) identifying expression vectors, or the DNA insert found in the expression vector, that are present in the reserved aliquot and not in cells maintaining the phenotype by subtraction means whereby expression vectors are identified that have targeted genes that affect the phenotype.    
     
     
         6 . The method of  claim 5  wherein the expression vector comprises a cDNA which is found in antisense orientation and which expresses antisense RNA.  
     
     
         7 . The method of  claim 5  wherein the expression vector comprises dominant negative gene fragments.  
     
     
         8 . A method of treating auto-immune disease in a subject by administering to the subject a therapeutically effective amount of a compound which inhibits a gene in the Fas pathway.  
     
     
         9 . The method according to  claim 8 , wherein said administering step includes administering an effective amount of a compound which inhibits a gene selected from the group consisting essentially of casein kinase alpha 1, Nrf-2, basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III,mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1.  
     
     
         10 . The method according to  claim 8 , said administering step includes administering an effective amount of a compound selected from the group consisting essentially of dicumarol, sulfinpyrazone, Nrf2 inhibitor, CKI-7 and casein Kinase inhibitor.