Expression of the heterologous genes according to a targeted expression profile
AssigneeThe University of Edinburgh
InventorsAustin Gerard Smith, Peter Scott Mountford, Richard Frank Lathe
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Abstract
This invention relates to DNA constructs for inserting heterologous gene sequences into a host genome so as to obtain expression of the heterologous gene, to methods of inserting heterologous gene sequences into a host genome, and to organisms carrying modified host genomes. Specifically, the DNA constructs of this invention contain an expression unit of an internal ribosome binding site (IRES) coupled to a heterologous gene sequence. This expression unit is flanked at the 5' and 3' ends by DNA sequences that enable homologous recombination or integration of the construct with the DNA of a targeted host to obtain expression of the heterologous gene in the host.
Claims29
A method of inserting a gene coding sequence into a target endogenous gene in a eukaryotic cellular host cell genome and expressing said gene coding sequence, by transforming the host cell with a vector comprising a DNA construct, wherein the host cell is a mouse embryonic stem cell and the DNA construct comprises the elements:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are substantially homologous with separate sequences from the target endogenous gene and are of sufficient length to undergo homologous recombination with the host cell genome so as to insert the A-P-B-Q-C elements into the host cell genome;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
A method of inserting a gene coding sequence into a eukaryotic, cellular host cell genome and expressing said coding sequence under control of elements regulating expression of an endogenous gene in a donor cell genome, said donor cell being a different cell from said host cell, by allowing a DNA construct to undergo random integration into the host cell genome, wherein the host cell is a mouse embryonic stem cell and the DNA construct comprises the sequence:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are homologous with separate sequences from the same donor cell genome and comprise the elements regulating expression of the endogenous gene in the donor cell;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
A method for inserting a gene coding sequence into a target endogenous gene in a eukaryotic cellular host cell genome and expressing said gene coding sequence in vitro, by transforming the host cell with a vector comprising a DNA construct, wherein the host cell is an animal stem cell and the DNA construct comprises the element:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are homologous with separate sequences from the same donor cell genome and comprise the elements regulating expression of the endogenous gene in the donor cell;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
A method for inserting a gene coding sequence into a eukaryotic, cellular host cell genome and expressing said coding sequence in vitro under control of elements regulating expression of an endogenous gene in a donor cell genome, said donor cell being a different cell from said host cell, by allowing a DNA construct to undergo random integration into the host cell genome, wherein the host cell is an animal stem cell and the DNA construct comprises the sequence:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are homologous with separate sequences from the same donor cell genome and comprise the elements regulating expression of the endogenous gene in the donor cell;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
A method of inserting a gene coding sequence into a target endogenous gene in a eukaryotic cellular host cell genome and expressing said gene coding sequence, by transforming the host cell with a vector comprising a DNA construct, wherein the host cell is a fertilized non-human egg and the DNA construct comprises the elements:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are substantially homologous with separate sequences from the target endogenous gene and are of sufficient length to undergo homologous recombination with the host cell genome so as to insert the A-P-B-Q-C elements into the host cell genome;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
A method of inserting a gene coding sequence into a eukaryotic, cellular host cell genome and expressing said coding sequence under control of elements regulating expression of an endogenous gene in a donor cell genome, said donor cell being a different cell from said host cell, by allowing a DNA construct to undergo random integration into the host cell genome, wherein the host cell is a fertilized non-human egg and the DNA construct comprises the sequence:
- 5'X-A-P-B-Q-C-Y3'
- in which
- X and Y are homologous with separate sequences from the same donor cell genome and comprise the elements regulating expression of the endogenous gene in the donor cell;
- P is an internal ribosome entry site (IRES);
- Q is the gene coding sequence;
- and
- A, B and C are, separately, linker sequences or a covalent bond.
Dependencies are inferred from the claim text.