Directed switch-mediated DNA recombination

A method for directed switch-mediated recombination, the method comprising the steps of:

• a) introducing a targeting construct into a cell,
• wherein the targeting construct comprises a switch region, and a promoter (P1) operably linked to and 5′ of the switch region, and
• wherein the cell comprises a target locus comprising a switch region, a target sequence adjacent and 3′ of the switch region, and a promoter (P2) operably positioned within the target locus to provide transcription of the switch region and target sequence;
• and
• b) culturing the cell to allow transcription of the target locus and the targeting construct, thereby promoting recombination between the target locus switch region and the targeting construct switch region;
• and
• c) selecting a cell comprising a modified target locus comprising the targeting construct promoter P1, a switch region, and the targeting construct,
• wherein P1, the switch region, and the target sequence are operably linked and wherein the target sequence is under control of P1.

A method for directed switch-mediated recombination, the method comprising the steps of:

• a) introducing a targeting construct into a cell,
• wherein the targeting construct comprises a switch region, and a promoter (P1) operably linked to and 5′ of the switch region, and a modifying sequence operably linked to and 3′ of the switch region,
• wherein the cell comprises a target locus comprising a switch region, a target sequence adjacent and 3′ of the switch region, and a promoter (P2) operably positioned within the target locus to provide transcription of the switch region and target sequence;
• and
• b) culturing the cell to allow transcription of the target locus and the targeting construct, thereby promoting recombination between the target locus switch region and the targeting construct switch region;
• and
• c) selecting a cell comprising a modified target locus comprising the targeting construct promoter P2, a switch region, and the modifying sequence,
• wherein P2, the switch region, and the modifying sequence are operably linked.

A method for -producing an antibody by switch-mediated recombination, the method comprising the steps of:

• a) introducing a targeting construct into an antibody-expressing cell, wherein the targeting construct comprises a switch region (S2), a promoter operably linked to and 5′ of the switch region, and a modifying sequence operably linked to and 3′ of the switch region, and
• wherein the antibody heavy chain expressed by the cell is encoded by an antibody heavy chain locus comprising a promoter, an antibody heavy chain variable region operably linked to and 3′ of the promoter, a switch region (S2) adjacent and 3′ of the variable region, and an antibody heavy chain constant region adjacent and 3′ of the switch region,
• b) culturing the cell to allow antibody expression and transcription of the targeting construct, wherein switch-mediated recombination between said S1 and S2 is promoted and wherein said heavy chain locus constant region is replaced with the modifying sequence of the targeting construct;
• and
• c) selecting a cell comprising a modified heavy chain locus, the modified heavy chain locus comprising the heavy chain locus promoter, the heavy chain variable region, a switch region, and the modifying sequence of the targeting construct, wherein the heavy chain locus promoter, the variable region, the switch region, and the modifying sequence are operably linked.