Efficient construction of gene targeting vectors
First Claim
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1. A method for preparing gene targeting vectors in yeast, the method comprising:
- a) preparing a shuttle vector comprising a first yeast selectable marker, a bacterial selectable marker and a fragment of genomic DNA containing at least part of a gene to be targeted;
b) preparing a specific engineered fragment (SEF) comprising a marker cassette, the marker cassette comprising a second yeast selectable marker different from the first yeast selectable marker, a selectable marker capable of expression in mammalian embryonic stem cells said marker cassette being flanked on each side by mammalian gene-specific flanking sequences homologous to a portion of the gene to be targeted;
c) transforming yeast cells with the shuttle vector of step a) and with the SEF of step b), and allowing said shuttle vector and said SEF to recombine by homologous recombination;
d) selecting the transformed yeast cells for expression of said first and second yeast selectable markers; and
e) isolating the targeting vector produced by recombination between the shuttle vector and the SEF from the yeast cells selected in step d).
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Abstract
The present invention is directed to methods for producing or obtaining gene targeting constructs by way of homologous recombination in host cells and to targeting constructs produced by those methods. The invention is also directed to transgenic animals having targeted mutations introduced in to the cells of the animal using the targeting constructs of the invention.
31 Citations
26 Claims
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1. A method for preparing gene targeting vectors in yeast, the method comprising:
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a) preparing a shuttle vector comprising a first yeast selectable marker, a bacterial selectable marker and a fragment of genomic DNA containing at least part of a gene to be targeted; b) preparing a specific engineered fragment (SEF) comprising a marker cassette, the marker cassette comprising a second yeast selectable marker different from the first yeast selectable marker, a selectable marker capable of expression in mammalian embryonic stem cells said marker cassette being flanked on each side by mammalian gene-specific flanking sequences homologous to a portion of the gene to be targeted; c) transforming yeast cells with the shuttle vector of step a) and with the SEF of step b), and allowing said shuttle vector and said SEF to recombine by homologous recombination; d) selecting the transformed yeast cells for expression of said first and second yeast selectable markers; and e) isolating the targeting vector produced by recombination between the shuttle vector and the SEF from the yeast cells selected in step d). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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- 12. A targeting construct comprising a marker cassette, the marker cassette comprising a first yeast selectable marker and a mammalian cell selectable marker, said cassette being flanked on each side by DNA homologous to a gene to be targeted, said targeting construct further comprising a second yeast selectable marker different from the first yeast selectable marker and a bacterial selectable marker.
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19. A method of preparing gene targeting constructs in homologous recombination competent cells the method comprising the steps of:
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a) preparing a first DNA construct, the first DNA construct comprising all or part of a gene to be targeted; b) preparing a second DNA construct, the second DNA construct comprising a DNA for insertion into a site in a mammalian gene, said DNA for insertion sequence being flanked on both sides by gene-specific sequences homologous to a portion of the gene to be targeted and c) introducing into host cells competent to mediate homologous recombination the first and second DNA constructs; d) allowing the first and second DNA constructs to recombine via their homologous sequences thereby producing a targeting construct; and e) isolating the targeting construct produced in step d). - View Dependent Claims (20, 21, 22, 23, 24)
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Specification