Back to results
US 6,284,541 B1Granted
Positive-negative selection for homologous recombination
AssigneeRoche Diagnostics GmbH
InventorsJohannes Auer, Raimund Sprenger, Konrad Honold
Life of this patent
Estimating the term…
Abstract
The invention concerns a method for the introduction of a foreign DNA into the genome of a target cell by homologous recombination as well as for the homologous recombination of suitable DNA constructs.
Claims32
A method for introducing foreign DNA into a host cell by homologous recombination, comprising transfecting the host cell with a recombinant vector comprising:
- a) two flanking nucleotide sequences which are homologous to a target gene sequence in the genome of the host cell,
- b) a nucleotide sequence encoding a positive selection marker located between the two flanking nucleotide sequences,
- c) nucleotide sequence encoding a negative selection marker located outside the two flanking sequences, and
- d) each of b) and c) are operatively linked to an expression control sequence which is active in the host cell,
- wherein the negative selection marker comprises at least one nucleotide sequence encoding a polypeptide located on the cell surface, and wherein the negative selection marker is not expressed after a targeted integration of the vector into the genome of the cell by homologous recombination, and is expressed and presented on the cell surface after a random integration of the vector into the genome of the cell.
2 more elements
A recombinant vector comprising
- a) two flanking nucleotide sequences that are homologous to a target gene sequence in a cell,
- b) a nucleotide sequence encoding a positive selection marker under the control of an expression control sequence that is active in the cell, wherein the nucleotide sequence is located inside of the two flanking sequences according to (a), and
- c) a nucleotide sequence encoding a negative selection marker under the control of an expression control sequence that is active in the cell, wherein the nucleotide sequence of c) is located outside the flanking homologous nucleotide sequences according to (a), and an expression product of the nucleotide is a polypeptide located on the cell surface.
A recombinant vector comprising
- a) two flanking nucleotide sequences that are homologous to a target gene sequence in a cell,
- b) a nucleotide sequence encoding a positive selection marker under the control of an expression control sequence that is active in the cell, wherein the nucleotide sequence is located inside of the two flanking sequences according to (a), and
- c) a nucleotide sequence encoding a negative selection marker under the control of an expression control sequence that is active in the cell, wherein the nucleotide sequence of c) is located outside the flanking homologous nucleotide sequences according to (a), and an expression product of the nucleotide sequence is a receptor fragment containing an extracellular ligand binding domain and located on the cell surface, with the proviso that an intracellular domain of the receptor fragment is fully or partially deleted or modified, and wherein by deleting or modifying said intracellular domain of said receptor fragment, said receptor fragment is incapable of signal transduction.
A method for endogenous gene activation comprising introducing foreign DNA into a host cell by homologous recombination, comprising transfecting the host cell with a recombinant vector comprising:
- a) two flanking nucleotide sequences which are homologous to a target gene sequence in the genome of the host cell,
- b) a nucleotide sequence encoding a positive selection marker located between the two flanking nucleotide sequences,
- c) a nucleotide sequence encoding a negative selection marker located outside the two flanking sequences, and
- d) each of b) and c) are operatively linked to a heterologous expression control sequence which is active in the host cell, and
- e) an additional heterologous expression control sequence which is active in the host cell, and which is between the two flanking homologous nucleotide sequences,
- wherein the negative selection marker comprises at least one nucleotide sequence encoding a polypeptide located on the cell surface, and wherein the negative selection marker is not expressed after a targeted integration of the vector into the genome of the cell by homologous recombination and is expressed and presented on the cell surface after a random integration of the vector into the genome of the cell.
3 more elements
Dependencies are inferred from the claim text.