Methods and vectors for making knockout animals
AssigneePurdue Research Foundation
InventorsPaul Collodi, Lianchun Fan
Life of this patent
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Abstract
The present invention includes methods for integrating a modifying polynucleotide into a target polynucleotide in a cell, or instance, a fish cell or a mouse cell, and methods for making chimeric animals, heterozygous animals, and homozygous animals that are chimeric, heterozygous, or homozygous, respectively, for a modifying polynucleotide integrated into a target polynucleotide. The present invention also includes vectors and methods of using the vectors for integrating a modifying polynucleotide into a target polynucleotide in a cell, for instance, a fish cell or a mouse cell.
Claims26
A method for integrating a polynucleotide comprising:
- providing a fish cell comprising a target polynucleotide, wherein the fish cell will become a germ cell when introduced to a fish embryo;
- introducing to the fish cell a modifying polynucleotide comprising a first homologous region and a second homologous region, wherein the first homologous region will undergo homologous recombination with a first region of the target polynucleotide, and wherein the second homologous region will undergo homologous recombination with a second region of the target polynucleotide, and
- identifying a recipient fish cell comprising the modifying polynucleotide integrated in the target polynucleotide.
A method for making a germ-line chimeric fish comprising:
- providing a fish cell comprising a modifying polynucleotide integrated in a target polynucleotide;
- introducing the fish cell to a recipient fish embryo to result in a chimeric fish embryo;
- and
- incubating the chimeric fish embryo to produce a chimeric fish comprising a germ cell derived from the introduced fish cell.
A method for making a fish heterozygous for a mutation comprising:
- providing a first gamete obtained from a chimeric fish, wherein the first gamete comprises a modifying polynucleotide integrated in a target polynucleotide;
- fertilizing the first gamete with a second gamete to result in a fertilized gamete, wherein the second gamete does not comprise the modifying polynucleotide integrated in the target polynucleotide;
- and
- incubating the fertilized gamete to produce a fish heterozygous for the modifying polynucleotide integrated in the target polynucleotide.
A method for making a fish homozygous for a mutation comprising:
- providing a first gamete obtained from a first fish, wherein the first gamete comprises a modifying polynucleotide integrated in a target polynucleotide;
- providing a second gamete obtained from a second fish, wherein the second gamete comprises the same modifying polynucleotide integrated in the same target polynucleotide as the first gamete;
- fertilizing the first gamete with the second gamete to result in a fertilized gamete;
- and
- incubating the fertilized gamete to produce a fish homozygous for the modifying polynucleotide integrated in the target polynucleotide.
A vector comprising a first homologous region, a second homologous region, and a first coding sequence encoding a selectable marker located between the first homologous region and second homologous region, wherein the first homologous region will undergo homologous recombination with a first region of a target polynucleotide present in a cell, and wherein the second homologous region will undergo homologous recombination with a second region of the target polynucleotide, the vector further comprising a second coding sequence encoding a detectable marker, the second coding sequence not located between the first homologous region and second homologous region.
A method for integrating a polynucleotide comprising:
- providing a cell comprising a target polynucleotide, wherein the cell will become a germ cell when introduced to an embryo;
- introducing to the cell a modifying polynucleotide comprising a first homologous region, a second homologous region, and a first coding sequence encoding a selectable marker located between the first homologous region and second homologous region, wherein the first homologous region will undergo homologous recombination with a first region of a target polynucleotide present in the cell, and wherein the second homologous region will undergo homologous recombination with a second region of the target polynucleotide, the modifying polynucleotide further comprising a second coding sequence encoding a detectable marker, the second coding sequence not located between the first homologous region and second homologous region;
- selecting for recipient cells expressing the selectable marker;
- and
- identifying a recipient cell that does not express the detectable marker, wherein expression of the selectable marker and absence of expression of the detectable marker indicates the cell comprises the modifying polynucleotide integrated in the target polynucleotide.
Dependencies are inferred from the claim text.