Directed switch-mediated DNA recombination

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

• introducing a targeting construct into an isolated B cell or B cell hybridoma,
• wherein the targeting construct comprises a first switch region, and a promoter (P.sub.1) operably linked to and 5' of the first switch region, and
• wherein the cell comprises a target locus comprising a second switch region, a target sequence adjacent and 3' of the second switch region, and a promoter (P.sub.2) operably positioned with in the target locus to provide transcription of the second switch region and target sequence;
• and
• culturing the cell to allow transcription of the first and second switch regions, thereby promoting recombination between the first switch region of the targeting construct and the second switch region of the target locus;
• and
• selecting a cell comprising a modified target locus comprising P.sub.1, a switch region, and the target sequence;
• wherein P.sub.1, the switch region, and the target sequence are operably linked and wherein the target sequence is under control of P.sub.1, indicating that directed switch-mediated recombination has occurred.

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

• introducing a targeting construct into an isolated B cell or B cel hybridoma that facilitates switch-mediated recombination when two switch regions are transcribed,
• where in the targeting construct comprises a first switch region, and a promoter (P.sub.1) operably linked to and 5' of the first switch region, and a modifying sequence operably linked to and 3' of the first switch region,
• wherein the cell comprises a target locus comprising a second switch region, a target sequence adjacent and 3' of the second switch region, and a promoter (P.sub.2) operably positioned within the target locus to provide transcription of the second switch region and target sequence;
• and
• culturing the cell to allow transcription of the first and second switch sequences, thereby promoting recombination between the first switch region of the targeting construct and the second switch region of the target locus;
• and
• selecting a cell comprising a modified target locus comprising P.sub.2, a switch region, and the modifying sequence;
• wherein P.sub.2, the switch region, and the modifying sequence are operably linked, indicating that directed switch-mediated recombination has occurred.

A method for producing a modified antibody heavy chain by switch-mediated recombination, the method comprising the steps of:

• a) introducing a targeting construct into an isolated B cell or B cell hybridoma that facilitates switch-mediated recombination, wherein the targeting construct comprises a switch region (S.sub.1), a promoter operably linked to and 5' of S.sub.1, and a modifying sequence operably linked to and 3' of S.sub.1, and
• wherein an 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 (S.sub.2) adjacent and 3' of the variable region, and an antibody heavy chain constant region adjacent and 3' of S.sub.2 ;
• b) culturing the cell to allow transcription of the targeting construct, wherein switch-mediated recombination between said S.sub.1 and S.sub.2 is promoted;
• 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;
• wherein a modified antibody heavy chain is produced.