Compositions and methods for mammalian genetics and uses thereof

Abstract

The invention provides compositions and methods for performing mammalian cell genetics, e.g., genetic screens, using near-haploid cells. The invention further provides genes and gene products isolated using the inventive methods and methods of use thereof.

1 . A method of identifying a gene that affects cell phenotype, the method comprising steps of: (a) introducing a gene trap vector into near-haploid mammalian cells in culture, wherein said gene trap vector comprises a nucleic acid construct that integrates into the genome of said near-haploid mammalian cell, and wherein the nucleic acid construct comprises a nucleic acid that allows the identification of a cell containing said nucleic acid; (b) identifying a cell containing said gene trap vector integrated into its genome, wherein the cell exhibits a phenotype of interest; and (c) identifying a gene into which the nucleic acid construct integrated, thereby identifying a gene that affects cell phenotype. 
     
     
         2 . The method of  claim 1 , wherein the nucleic acid encodes a reporter that allows the identification of a cell expressing the nucleic acid. 
     
     
         3 . The method of  claim 1  or  2 , wherein the near-haploid mammalian cell is a human cell, a KBM7 cell, or is genetically modified. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The method of any one of  claims 1  to  3 , wherein the nucleic acid construct comprises in operable association in a 5′ to 3′ direction: (1) a splice acceptor site; (2) a nucleic acid encoding a reporter that allows the identification of a cell expressing the nucleic acid; and (3) a polyadenylation sequence. 
     
     
         7 . The method of  claim 1 , wherein the splice acceptor site is an adenoviral splice acceptor site. 
     
     
         8 . The method of any one of  claims 1  to  3  or  6  to  7 , wherein the phenotype of interest is altered susceptibility to infection by a pathogen as compared with susceptibility of a suitable control cell to the pathogen. 
     
     
         9 . The method of  claim 8 , wherein the method comprises identifying cells that express the reporter and are resistant to the pathogen. 
     
     
         10 . The method of any one of  claims 1  to  3  or  6  to  7 , wherein the phenotype of interest is altered sensitivity to a compound of interest as compared with sensitivity of suitable control cell to the compound. 
     
     
         11 . The method of  claim 10 , wherein the compound of interest is a therapeutic agent, a therapeutic agent used to treat cancer, a cytotoxic agent, or a bacterial toxin. 
     
     
         12 .- 14 . (canceled) 
     
     
         15 . The method of  claim 10 , wherein the compound of interest is a toxin and the method comprises identifying cells that express the reporter and are resistant to the toxin. 
     
     
         16 . The method of any one of  claims 1  to  3  or  6  to  7 , wherein the phenotype of interest is altered propensity to undergo apoptosis as compared with propensity of a suitable control cell to undergo apoptosis. 
     
     
         17 . The method of any one of  claims 1  to  3 ,  6  to  11  or  15  to  16 , wherein step (b) comprises recovering and sequencing a portion of the gene. 
     
     
         18 . The method of any one of  claims 1  to  3 ,  6  to  11  or  15  to  17 , wherein the near-haploid mammalian cell further comprises a reporter useful to identify cells having a phenotype of interest. 
     
     
         19 . The method of any one of  claims 1  to  3 ,  6  to  11  or  15  to  18 , wherein step (a) comprises introducing the gene trap vector into cells of a near-haploid mammalian cell line, wherein the gene trap vector comprises a nucleic acid construct comprising a nucleic acid encoding a reporter that allows the identification of cells expressing said nucleic acid, wherein said nucleic acid construct integrates into the genome of at least some of said near-haploid mammalian cells; (b) identifying a plurality of cells containing said gene trap vector so integrated, wherein the cells exhibit a phenotype of interest; and (c) identifying a plurality of genes into which the nucleic acid construct integrated, thereby identifying a plurality of genes that affect cell phenotype. 
     
     
         20 . A method of identifying a gene that encodes a host cell factor that affects susceptibility to a pathogen, the method comprising steps of: (a) introducing a gene trap vector into near-haploid mammalian cells, wherein the gene trap vector comprises a nucleic acid construct comprising a nucleic acid encoding a reporter that allows the identification of a cell expressing said nucleic acid, wherein said nucleic acid construct integrates into the genome of said haploid mammalian cell; (b) contacting the near-haploid mammalian cells with a pathogen or virulence factor; (c) identifying a cell that contains said nucleic acid construct integrated into its genome and exhibits altered susceptibility to the pathogen or virulence factor; and (d) identifying a gene into which the nucleic acid construct integrated, thereby identifying a gene that encodes a host cell factor that affects susceptibility to a pathogen. 
     
     
         21 . The method of  claim 20 , wherein step (b) comprises identifying a cell that is resistant to the pathogen or virulence factor. 
     
     
         22 . A method of identifying a gene that encodes a gene product that plays a role in activity of an agent in mammalian cells, the method comprising steps of: (a) introducing a gene trap vector into near-haploid mammalian cells, wherein the gene trap vector comprises a nucleic acid construct comprising a nucleic acid encoding a reporter that allows the identification of a cell expressing said nucleic acid, wherein said nucleic acid construct integrates into the genome of at least some of said near-haploid mammalian cells; (b) contacting the mammalian cells with an agent at a concentration sufficient to cause a detectable effect on non-mutant near-haploid cells; (c) identifying a cell that contains said nucleic acid construct integrated into its genome and does not exhibit said effect; and (d) identifying a gene into which the nucleic acid construct integrated, thereby identifying a gene that encodes a gene product that plays a role in activity of the agent in mammalian cells. 
     
     
         23 . The method of  claim 22 , wherein the agent is a drug. 
     
     
         24 .- 49 . (canceled)