Compositions and methods for making mutations in cell lines and animals

A method for mutating a gene in a cell comprising:

• subjecting a cell first to insertional mutagenesis and then to physicochemical mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated.

A method for mutating a gene in a cell comprising:

• subjecting a cell to physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated.

A method for mutating a gene in a cell and identifying the mutated gene comprising:

• subjecting a cell to simultaneous physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated and identifying the gene by means of the insertional mutagen.

A method for mutating a gene in a cell and identifying the mutated gene comprising:

• subjecting a cell first to insertional mutagenesis and then to physicochemical mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated and identifying the gene by means of the insertional mutagen.

A method for mutating a gene in a cell and identifying the mutated gene comprising:

• subjecting a cell to physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated and identifying the gene by means of the insertional mutagen.

A method for mutating two or more genes in a cell, wherein the mutations are required cumulatively to produce a phenotype, comprising:

• subjecting a cell 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.

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, comprising:

• subjecting a cell 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 and identifying at least one insertionally mutated gene by means of the insertional mutagen.

A method for correlating a phenotype of a cell with a mutated gene comprising:

• simultaneously physicochemically and insertionally mutagenizing a cell, screening the cell for a desired phenotype, and identifying the mutated gene that correlates with the phenotype by means of the insertional mutagen.

A method for correlating a phenotype of a cell with a mutated gene comprising:

• physicochemically and insertionally mutagenizing a cell, screening the cell for a desired phenotype, and identifying the mutated gene that correlates with the phenotype by means of the insertional mutagen, wherein insertional mutagenesis precedes physico-chemical mutagenesis.

A method for correlating a phenotype of a cell with a mutated gene comprising:

• physicochemically and insertionally mutagenizing a cell, screening the cell for a desired phenotype, and identifying the mutated gene that correlates with the phenotype by means of the insertional mutagen.

A method for selecting a mutated cell from a plurality of cells comprising:

• subjecting a plurality of cells to physicochemical and insertional mutagenesis and selecting an insertionally mutated cell by means of the insertional mutagen.

A method for creating a homozygous mutation in a multicellular organism comprising:

• subjecting a first organism to physicochemical mutagenesis, subjecting an organism of the opposite sex to the first organism to insertional mutagenesis, breeding the first and second mutated organisms, and obtaining progeny from the breeding wherein the progeny is homozygous for a mutation in a gene wherein one allele of the gene is mutated by physciochemical mutagenesis and the second allele of the gene is mutated by insertional mutagenesis.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by a physico-chemical mutagen, said method comprising subjecting said cell to an insertional mutagen such that the second allele of said gene is insertionally mutated.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by an insertional mutagen, said method comprising subjecting said cell to a physico-chemical mutagen such that the second allele of said gene is physico-chemically mutated.

A method for mutating two or more genes in a cell where said mutations are required cumulatively to produce a phenotype, said method comprising subjecting a cell to physico-chemical mutagenesis such that a sufficient number of genes is physico-chemically mutated to produce said phenotype, said cell containing an insertional mutation in at least one of said genes required to produce said phenotype.

A method for mutating two or more genes in a cell and identifying at least one of the mutated genes, where said mutations are required cumulatively to produce a phenotype, said method comprising subjecting a cell to physico-chemical mutagenesis such that a sufficient number of genes is physico-chemically mutated to produce said phenotype, said cell containing an insertional mutation in at least one of said genes required to produce said phenotype, and identifying at least one insertionally-mutated gene by means of the insertional mutagen.

A cell in vitro where one allele of a gene in said cell is insertionally mutated and the second allele of said gene is physicochemically mutated.

A cell containing a mutation in two or more genes where at least one of the mutations is an insertional mutation, wherein the mutations are cumulatively required to produce a phenotype in said cell.

A library of two or more physicochemically mutated cells, one or more of which cells has been contacted with one or more insertional mutagens, wherein one or more insertional mutagens has been inserted into the genome of at least one cell within said library.

A library of one or more insertionally mutated cells each comprising one or more insertional mutagens inserted into the genome of said cell, wherein one or more cells from the library has been treated with one or more physicochemical mutagens.

A cell in vitro that is homozygous for at least one mutation in an endogenous gene, wherein said cell comprises an insertional mutation in a first allele of said gene and a non-insertional mutation in a second allele of said gene.

A cell library comprising 1-106 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A cell library comprising 1-105 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A cell library comprising 1-10 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A cell library comprising 50-100 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A cell library comprising about 30-about 400 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A cell library comprising 2, 5, 10, 25, 50, 100, 200, 400, 600 or 800 physicochemically mutated clones wherein one of more of said physicochemically mutagenized clones has been subjected to insertional mutagenesis.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising 1-106 physicochemically mutated clones.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising 1-105 physicochemically mutated clones.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising 1-103 physicochemically mutated clones.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising 50-100 physicochemically mutated clones.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising about 30-about 400 physicochemically mutated clones.

A method for creating a mutagenized cell library, said method comprising insertionally mutagenizing a cell library comprising 2, 5, 10, 25, 50, 100, 200, 400, 600 or 800 physicochemically mutated clones.

A method for mutating a gene in a cell comprising:

• subjecting a cell to physiochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A method for mutating a gene in a cell comprising:

• subjecting a cell to physiochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by a physicochemical mutagen, said method comprising subjecting said cell to an insertional mutagen such that the second allele of said gene is insertionally mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by a physicochemical mutagen, said method comprising subjecting said cell to an insertional mutagen such that the second allele of said gene is insertionally mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by an insertional mutagen, said method comprising subjecting said cell to a physicochemical mutagen such that the second allele of said gene is physicochemically mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A method for mutating a gene in a cell, said cell containing a mutation in one allele of said gene induced by an insertional mutagen, said method comprising subjecting said cell to a physicochemical mutagen such that the second allele of said gene is physicochemically mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A cell in vitro where one allele of a gene in said cell is insertionally mutated and the second allele of said gene is physicochemically mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A cell in vitro where one allele of a gene in said cell is insertionally mutated and the second allele of said gene is physicochemically mutated, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A library of two or more physicochemically mutated cells, one or more of which cells has been contacted with one or more insertional mutagens, wherein one or more insertional mutagens has been inserted into the genome of at least one cell within said library, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A library of two or more physicochemically mutated cells, one or more of which cells has been contacted with one or more insertional mutagens, wherein one or more insertional mutagens has been inserted into the genome of at least one cell within said library, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A library of one or more insertionally mutated cells each comprising one or more insertional mutagens inserted into the genome of said cell, wherein one or more cells from the library has been treated with one or more physicochemical mutagens, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A library of one or more insertionally mutated cells each comprising one or more insertional mutagens inserted into the genome of said cell, wherein one or more cells from the library has been treated with one or more physicochemical mutagens, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A cell in vitro that is homozygous for at least one mutation in an endogenous gene, wherein said cell comprises an insertional mutation in a first allele of said gene and a non-insertional mutation in a second allele of said gene, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A cell in vitro that is homozygous for at least one mutation in an endogenous gene, wherein said cell comprises an insertional mutation in a first allele of said gene and a non-insertional mutation in a second allele of said gene, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.

A method for mutating a gene in a cell comprising:

• subjecting a cell to physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated wherein said cell is screened for a desired phenotype following physicochemical mutagenesis and before insertional mutagenesis.

A method for mutating a gene in a cell and identifying the mutated gene comprising:

• subjecting a cell to physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated and identifying the gene by means of the insertional mutagen, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor operably-linked to a promoter in the polynucleotide.

A method for mutating a gene in a cell and identifying the mutated gene comprising:

• subjecting a cell to physicochemical mutagenesis and insertional mutagenesis such that one allele of said gene is physicochemically mutated and the other allele is insertionally mutated and identifying the gene by means of the insertional mutagen, wherein the allele that is insertionally mutated is insertionally mutated using a polynucleotide that does not comprise an unpaired splice donor.