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US 2004/0170614 A1Published application
Somatic cell gene targeting vectors and methods of use thereof
OwnerGail Bishop, Bruce S. Hostagerinventors
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Abstract
The present invention provides vectors for gene targeting and disruption in somatic cells, and methods for using the same.
Claims31
A somatic cell gene targeting vector comprising:
- (a) a gene targeting construct comprising a first cloning site operably linked to a DNA encoding a positive selection marker, a second cloning site and a first polyadenylation sequence, wherein the construct is promoterless;
- and
- (b) an expression cassette comprising a promoter operably linked to DNA encoding a negative selection marker and a second polyadenylation sequence.
A method for disrupting a gene of interest in a somatic cell, which method comprises introducing a targeting vector comprising a gene targeting construct comprising a first cloning site operably linked to a DNA encoding a positive selection marker, a second cloning site and a first polyadenylation sequence, wherein the construct is promoterless;
- and an expression cassette comprising a promoter operably linked to DNA encoding a negative selection marker and a second polyadenylation sequence, wherein the first cloning site comprises a first DNA segment that is homologous to a first genomic target sequence and the second cloning site comprises a second DNA segment that is homologous to a second genomic target sequence, into a somatic cell such that the first genomic target sequence and the second genomic target sequence recombine with the gene to yield a genetically altered cell.
A method for disrupting a gene of interest in a somatic cell, which method comprises:
- (a) introducing a targeting vector comprising a gene targeting construct comprising a first cloning site operably linked to a DNA encoding a positive selection marker, a second cloning site and a first polyadenylation sequence, wherein the construct is promoterless;
- and an expression cassette comprising a promoter operably linked to DNA encoding a negative selection marker and a second polyadenylation sequence, wherein the first cloning site comprises a first DNA segment that is homologous to a first genomic target sequence and the second cloning site comprises a second DNA segment that is homologous to a second genomic target sequence, into the somatic cell such that the first genomic target sequence and the second genomic target sequence recombine with the gene to yield a first genetically altered cell;
- and
- (b) introducing a recombinase to the first genetically altered cell, such that the positive selection marker is removed from the construct to yield a second genetically altered cell.
1 more element
Dependencies are inferred from the claim text.