Genome mutation by intron insertion into an embryonic stem cell genome
First Claim
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1. An in vitro method of creating a mutation in a genomic exon in a genome of a mouse embryonic stem (ES) cell, comprising:
- a) constructing a targeting vector comprising(i) a 5′
homology arm,(ii) an intron, wherein the intron comprising a drug selection cassette, wherein the drug selection cassette consists of a drug resistance gene operably linked to a promoter and flanked by a first and a second site-specific recombination sites, and(iii) a 3′
homology arm, wherein the 5′ and
3′
homology arms contain sequences homologous to corresponding genomic sequences; and
b) introducing the targeting vector into the ES cell such that the exon is mutated by the introduction of the intron.
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Abstract
Methods of creating mutations in genomic exons by inserting introns into the genomic exons via homologous recombination. Also, methods are provided for introducing modifications into genomic exons by inserting introns into the genomic exons via homologous recombination such that a mature mRNA transcript produced from a genomic region of the genome comprising the genomic exon does not contain the modification are provided. The methods provide for a rapid method for introducing mutations and/or modifications of any type into a mammalian cell genome.
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9 Claims
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1. An in vitro method of creating a mutation in a genomic exon in a genome of a mouse embryonic stem (ES) cell, comprising:
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a) constructing a targeting vector comprising (i) a 5′
homology arm,(ii) an intron, wherein the intron comprising a drug selection cassette, wherein the drug selection cassette consists of a drug resistance gene operably linked to a promoter and flanked by a first and a second site-specific recombination sites, and (iii) a 3′
homology arm, wherein the 5′ and
3′
homology arms contain sequences homologous to corresponding genomic sequences; andb) introducing the targeting vector into the ES cell such that the exon is mutated by the introduction of the intron. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification