Directed switch-mediated DNA recombination

Abstract

Switch regions derived from an immunoglobulin (Ig) gene are used to direct recombination between a targeting construct containing a promoter, a switch region (S 1 ), and 2) a target locus minimally containing a promoter, a switch region (S 2 ), and a target sequence.

What is claimed is:  
     
         1 . An artificial nucleic acid targeting construct comprising: 
 a) a switch region; and    b) a promoter operably linked to and 5′ of the switch region.    
     
     
         2 . The targeting construct of  claim 1  further comprising a modifying sequence operably linked to and 3′ of the switch region.  
     
     
         3 . The targeting construct of  claim 1 , further comprising an additional DNA sequence, said additional DNA sequence positioned 5′ of the switch region and 3′ of the promoter.  
     
     
         4 . The targeting construct of  claim 2 , wherein the modifying sequence encodes a constant region of an antibody heavy chain.  
     
     
         5 . The targeting construct of  claim 4 , wherein the constant region is the constant region of a human antibody heavy chain.  
     
     
         6 . The targeting construct of  claim 4 , wherein the modifying sequence encodes a constant region of a murine antibody heavy chain.  
     
     
         7 . The targeting construct of  claim 4 , wherein the constant region is selected from the group consisting of Cγ, Cμ, Cα, Cδ, and Cε.  
     
     
         8 . The targeting construct of  claim 2 , wherein the modifying sequence encodes an immunoglobulin variable region, a promoter, an enzyme, a toxin, a hormone, a growth factor, a detectable peptide label, or a linker polypeptide.  
     
     
         9 . The targeting construct of  claim 1 , wherein the promoter is a constitutive or inducible promoter.  
     
     
         10 . The targeting construct of  claim 9 , wherein the promoter is a cytomegalovirus promoter, a spleen-focus forming virus (SFFV), a  Rous sarcoma  virus promoter, and SV40 promoter or a murine Maloney virus promoter.  
     
     
         11 . A cell comprising the targeting construct of  claim 1 .  
     
     
         12 . A cell line comprising a genome which has been subjected to directed switch-mediated recombination via an exogenous nucleic acid targeting construct, said targeting construct comprising a switch region and a promoter operably linked to and 5′ of the switch region.  
     
     
         13 . The cell line of  claim 12 , wherein said targeting construct further comprises a modifying sequence operably linked to and 3′ of the switch region.  
     
     
         14 . The cell line of  claim 13 , wherein said modifying sequence encodes a constant region of an antibody heavy chain.  
     
     
         15 . The cell line of  claim 14 , wherein said antibody heavy chain is a human antibody heavy chain.  
     
     
         16 . A recombinant protein produced by a cell line, said cell line comprising a genome which has been subjected to directed switch-mediated recombination via an exogenous nucleic acid targeting construct, said targeting construct comprising a switch region and a promoter operably linked to and 5′ of the switch region.  
     
     
         17 . The protein of  claim 16 , wherein said targeting construct further comprises a modifying sequence operably linked to and 3′ of the switch region.  
     
     
         18 . The protein of  claim 17 , wherein said modifying sequence encodes a constant region of an antibody heavy chain.  
     
     
         19 . The cell line of  claim 18 , wherein said antibody heavy chain is a human antibody heavy chain.  
     
     
         20 . 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 (P 1 ) 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 (P 2 ) 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 P 1 , a switch region, and the targeting construct, 
 wherein P 1 , the switch region, and the target sequence are operably linked and wherein the target sequence is under control of P 1 .  
   
     
     
         21 . 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 (P 1 ) 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 (P 2 ) 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 P 2 , a switch region, and the modifying sequence, 
 wherein P 2 , the switch region, and the modifying sequence are operably linked.  
   
     
     
         22 . The method of  claim 21 , wherein the target locus encodes an antibody heavy chain.  
     
     
         23 . The method of  claim 21 , wherein the target locus encodes a murine antibody heavy chain and the modifying sequence of the target construct is a human antibody heavy chain constant region.  
     
     
         24 . The method of  claim 21 , wherein the target locus further comprises a non-target sequence positioned between the target locus promoter and the target locus switch region.  
     
     
         25 . The method of  claim 24 , wherein the non-target sequence encodes a human antibody heavy chain variable region, the target sequence encodes a murine antibody heavy chain constant region, and the modifying sequence of the targeting construct is a human antibody heavy chain constant region.  
     
     
         26 . The method of  claim 21 , wherein the targeting construct is linearized prior to step a), and a cell containing a stable integrant of the targeting construct is selected prior to step b).  
     
     
         27 . The method of  claim 21 , wherein the targeting construct is extrachromosomal.  
     
     
         28 . 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 (S 2 ), 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 (S 2 ) 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 S 1  and S 2  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.    
     
     
         29 . A nucleic acid target locus comprising: a switch region, a first cloning site for insertion of a non-target sequence, the first cloning site being adjacent and 5′ of the switch region, a second cloning site for inserting of a target sequence, the second cloning site being adjacent and 3′ of the switch region, and a promoter operably linked to and 5′ of the first cloning site, wherein transcriptional activation of the promoter provides transcription of the non-target sequence, the switch region, and the target sequence.  
     
     
         30 . The construct of  claim 29 , wherein the switch region, first cloning site, second cloning site and promoter are operatively linked in a plasmid.