Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

Abstract

A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

1 - 41 . (canceled) 
     
     
         42 . A method for selecting a mammalian cell that has high expression of an immunoglobulin of interest comprising:
 (i) transfecting mammalian cells with a marking vector, wherein said marking vector comprises
 (a) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination, 
 (b) a first DNA fragment encoding a selectable marker protein that provides for selection of mammalian cells which have been successfully integrated with the marking vector wherein the selectable marker protein is not a neomycin phosphotransferase, and 
 (c) a second DNA fragment comprising at least one, but not all, exons of a neomycin phosphotransferase gene; 
   (ii) selecting in vitro for the mammalian cells which contain a single marking vector integrated into the genome wherein said cells have high expression of the selectable marker;   (iii) transfecting the mammalian cells that have high expression of the selectable marker with a targeting vector comprising
 (a) a third DNA fragment comprising the remaining neomycin phosphotransferase exon or exons that are not present the marking vector, 
 (b) an immunoglobulin DNA that encodes at least one immunoglobulin of interest, and 
 (c) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site; and 
   (iv) selecting in vitro for the mammalian cells which express said selectable marker, said neomycin phosphotransferase and said immunoglobulin of interest; thereby selecting a mammalian cell that has high expression of said immunoglobulin of interest.   
     
     
         43 . The method of  claim 42 , wherein the selectable marker DNA encodes a protein that is selected from the group consisting of histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase. 
     
     
         44 . The method of  claim 43 , wherein the selectable marker DNA encodes a dihydrofolate reductase protein. 
     
     
         45 . The method of  claim 42 , which further comprises determining the RNA levels of the selectable marker. 
     
     
         46 . The method of  claim 42 , wherein said second DNA fragment encoding the remaining neomycin phosphotransferase exons comprise two neomycin phosphotransferase exons which are separated by the immunoglobulin DNA that encodes at least one immunoglobulin of interest. 
     
     
         47 . The method of  claim 42 , wherein said immunoglobulin DNA encodes a heavy chain and a light chain. 
     
     
         48 . A eukaryotic cell which comprises a marking vector integrated into the genome for identifying a transcriptionally active site in the genome of a mammalian cell, wherein said marking vector comprises:
 (i) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination,   (ii) a first selectable marker DNA encoding for a first selectable marker that provides for selection of mammalian cells which have been successfully integrated with the marking vector, and   (iii) a first DNA fragment encoding a portion of a second selectable marker that is different from the first selectable marker, wherein said second selectable marker is only expressed in said mammalian cell when a targeting vector comprising (a) a second DNA fragment encoding a second portion of said second selectable marker DNA and (b) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site.   
     
     
         49 . The eukaryotic cell of  claim 48 , wherein said cell is a mammalian cell. 
     
     
         50 . The eukaryotic cell of  claim 49 , wherein said cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, myeloma cells, baby hamster kidney cells, COS cells, NSO cells, HeLa cells, and NIH 3T3 cells. 
     
     
         51 . The eukaryotic cell of  claim 50 , wherein the cell is a CHO cell. 
     
     
         52 . The eukaryotic cell of  claim 51 , wherein the cell is clone 15C9. 
     
     
         53 . A method for marking a transcriptionally active site in the genome of a mammalian cell comprising:
 (i) transfecting said mammalian cell with a marking vector, wherein said marking vector comprises (a) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination, (b) a first selectable marker DNA encoding for a first selectable marker that provides for selection of mammalian cells which have been successfully integrated with the marking vector, and (c) a first DNA fragment encoding a portion of a second selectable marker that is different from the first selectable marker, wherein said second selectable marker is only expressed in said mammalian cell when a targeting vector comprising (a) a second DNA fragment encoding a second portion of said second selectable marker and (b) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site; and   (ii) selecting in vitro for the mammalian cells which contain a single marking vector integrated into the genome wherein said cells have high expression of said first selectable marker; thereby marking the transcriptionally active site in the genome of said mammalian cell.   
     
     
         54 . The method of  claim 53 , wherein the in vitro screening comprises determining the protein levels of the first selectable marker. 
     
     
         55 . The method of  claim 54 , wherein the in vitro screening further comprises determining the RNA levels of the first selectable marker. 
     
     
         56 . The method of  claim 53 , wherein the first selectable marker DNA encodes a protein that is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase. 
     
     
         57 . The method of  claim 56 , wherein the first selectable marker DNA encodes a dihydrofolate reductase protein. 
     
     
         58 . The method of  claim 53 , wherein the second selectable marker DNA encodes a protein that is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase. 
     
     
         59 . The method of  claim 58 , wherein the second selectable marker DNA encodes a neomycin phosphotransferase protein. 
     
     
         60 . The method of  claim 53 , wherein the DNA region that is heterologous to the mammalian cell genome is a bacterial DNA, an insect DNA, a viral DNA or a synthetic DNA. 
     
     
         61 . The method of  claim 53 , wherein the region of DNA that is heterologous to the mammalian cell genome does not contain any functional genes.