Compositions and Methods for Making Mutations in Cell Lines and Animals

Abstract

The present invention is directed generally to reduction or inactivation of gene function or gene expression in cells in vitro and in multicellular organisms. The invention encompasses methods for mutating cells using a combination of mutagens, particularly wherein at least one mutagen is an insertional mutagen, to achieve homozygous gene mutation or mutation of multiple genes required cumulatively to achieve a phenotype to create knock-outs, knock-downs, and other modifications in the same cell. The invention is also directed to cells (and libraries thereof) and organisms created by the methods of the invention, including those in which at least one of the genes created by insertional mutagenesis is tagged by means of the insertion sequences thereby allowing identification of the mutated gene(s). The invention is also directed to libraries of mutated cells and their uses. The invention is also directed to methods of identifying mutations with methods of the invention, in cells (and libraries thereof) and organisms, by means of the insertional tag.

1 - 210 . (canceled) 
     
     
         211 . A method for mutating a gene in a cell, said method comprising:
 subjecting a cell in vitro to physicochemical mutagenesis with a physicochemical mutagen and insertional mutagenesis with a polynucleotide insertional mutagen such that one allele of said gene is physicochemically mutated and a second allele of said gene is insertionally mutated, wherein said insertional mutagen comprises a recombination sequence that allows excision of part or all of the insertional mutagen following insertion.   
     
     
         212 . A method for mutating a gene in a cell and identifying the mutated gene, said method comprising:
 subjecting a cell in vitro to physicochemical mutagenesis and insertional mutagenesis with a polynucleotide insertional mutagen such that one allele of said gene is physicochemically mutated and a second allele of said gene is insertionally mutated and identifying the gene by means of the insertional mutagen,   wherein the cell that is insertionally mutagenized is in a cell library comprising physicochemically-mutated clones wherein the mutation frequency of physicochemically-mutated alleles is about 1× genome coverage per 2-10 3  physiochemically-mutated clones, and wherein one or more of said physicochemically-mutated clones contains one or more insertional mutagens.   
     
     
         213 . A method for mutating two or more genes in a cell, wherein the mutations are required cumulatively to produce a phenotype, said method comprising:
 subjecting a cell in vitro to physicochemical mutagenesis and insertional mutagenesis such that said two or more genes are physicochemically mutated or insertionally mutated, but, at least one gene of the two or more genes is insertionally mutated, wherein said insertional mutagen comprises a recombination sequence that allows excision of part or all of the insertional mutagen following insertion.   
     
     
         214 . A method for mutating two or more genes in a cell and identifying at least one of the mutated genes, wherein the mutations are required cumulatively to produce a phenotype, said method comprising:
 subjecting a cell in vitro to physicochemical mutagenesis and insertional mutagenesis with a polynucleotide insertional mutagen such that said two or more genes are physicochemically mutated or insertionally mutated, but, at least one gene of the two or more genes is insertionally mutated, and identifying at least one insertionally mutated gene by means of the insertional mutagen,   wherein the cell that is insertionally mutagenized is in a cell library comprising physicochemically-mutated clones wherein the mutation frequency of physicochemically-mutated alleles is about 1× genome coverage per 2-10 3  physicochemically-mutated clones, and wherein one or more of said physicochemically-mutated clones contains one or more insertional mutagens.   
     
     
         215 . A method for correlating a phenotype of a cell with a mutated gene comprising:
 physicochemically mutagenizing and insertionally mutagenizing, with a polynucleotide insertional mutagen, a cell in vitro, screening the cell for a desired phenotype, and identifying the mutated gene that correlates with the phenotype by means of the insertional mutagen,   wherein the cell that is insertionally mutagenized is in a cell library comprising physicochemically-mutated clones wherein the mutation frequency of physicochemically-mutated alleles is about 1× genome coverage per 2-10 3  physicochemically-mutated clones, and wherein one or more of said physicochemically-mutated clones contains one or more insertional mutagens.   
     
     
         216 . The method of  claim 211 , wherein said physicochemical mutagen is selected from the group consisting of UV irradiation, gamma irradiation, x-rays, a restriction enzyme, a mutagenic or teratogenic chemical, a DNA repair mutagen, a DNA repair inhibitor, an error-prone DNA replication protein, N-ethyl-N-nitrosourea (ENU), ethylmethanesulphonate (EMS) and ICR191. 
     
     
         217 . The method of  claim 211 , wherein said insertional mutagen comprises one or more physical or functional nucleotide sequences selected from the group consisting of one or more multiple cloning sites, one or more transcription termination sites, one or more transcriptional regulatory sequences, one or more translational signal sequences, one or more open reading frames (ORFs), one or more sequences for mutating ORFs, one or more stop codons, one or more sequences for mutating or eliminating stop codons, one or more mRNA destabilizing elements, one or more hairpin sequences, one or more sequences for mutating or eliminating hairpins, one or more reporter genes, one or more splice acceptor sequences, one or more splice donor sequences, one or more internal ribosome entry sites (IRES), one or more transposon sequences, one or more site-specific recombination site sequences, one or more restriction enzyme sequences, one or more nucleotide sequences encoding a fusion partner protein or peptide, one or more selectable markers or selection modules, one or more bacterial sequences useful for propagating a vector in a host cell, one or more 3′ gene traps, one or more 5′ gene traps, one or more nucleotide sequences encoding localization signals, one or more origins of replication, nucleotide sequences encoding one or more protease cleavage sites, nucleotide sequences encoding one or more desired proteins or peptides encoded by a gene or a portion of a gene, and one or more sequences encoding one or more 5′ or 3′ polynucleotide tails. 
     
     
         218 . The method of  claim 217 , wherein said insertional mutagen comprises at least one splice acceptor site. 
     
     
         219 . The method of  claim 218 , wherein, upon integration of said insertional mutagen into the genome of said cell, said splice acceptor site is operably linked to a splice donor site of said gene. 
     
     
         220 . The method of  claim 211 , wherein said cell is contacted with said physicochemical mutagen and said insertional mutagen simultaneously. 
     
     
         221 . The method of  claim 211 , wherein said cell is contacted with said physicochemical mutagen and said mutagenic insertional mutagen sequentially. 
     
     
         222 . The method of  claim 221 , wherein said cell is contacted with said physicochemical mutagen before being contacted with said insertional mutagen. 
     
     
         223 . The method of  claim 221 , wherein said cell is contacted with said physicochemical mutagen after being contacted with said insertional mutagen. 
     
     
         224 . An isolated and cloned cell where one allele of a gene in said cell is insertionally mutated with a polynucleotide insertional mutagen and a second allele of said gene is physicochemically mutated. 
     
     
         225 . An isolated and cloned cell containing a mutation in two or more genes, wherein at least one of the mutations is by a polynucleotide insertional mutagen, and wherein the mutations are cumulatively required to produce a phenotype in said cell. 
     
     
         226 . A cell produced by the method of  claim 211 . 
     
     
         227 . A library of cells produced by the method of  claim 211 .