Mutant cell lines and methods for producing enhanced levels of recombinant proteins

Abstract

Mammalian somatic cells having a homozygous disruption in the gene which encodes the endoribonuclease known as RNase L and a homyzgous disruption in the gene which encodes the double-stranded RNA dependent kinase known as PKR. Methods for producing enhanced levels of recombinant proteins, or polypeptides, in mammalian cell systems are also provided. In one aspect the method employs cells having a homozygous disruption in the RNase L gene and a homozygous disruption PKR gene and comprises transfecting the cells with a nucleic acid, or polynucleotide, encoding a desired, exogenous protein, or polypeptide; expressing the exogenous protein in the cell; and isolating the exogenous protein from the transfected cells. In another aspect the method employs RNase L null cells transfected with a nucleic acid encoding a desired, exogenous protein. Preferably, the RNase L null cells are co-transfected with a nucleic acid encoding adenovirus VAI RNA, or a nucleic acid encoding a dominant negative PKR polypeptide, or a combination thereof. In another aspect the methods employ mutant cells having a homozygous disruption in the PKR gene, i.e. PKR null cells. In one embodiment, the method comprises co-transfecting PKR null cells with a nucleic acid encoding adenovirus VAI RNA and with a nucleic acid encoding a desired, exogenous protein, expressing the exogenous protein in the cell, and isolating the exogenous protein from the system. In another embodiment, the method comprises co-transfecting the PKR null cells with a nucleic acid encoding a dominant negative RNase L and with a nucleic acid encoding a desired, exogenous protein. In another aspect, the method comprises cotransfecting mammalian cells with a nucleic acid encoding the desired exogenous protein and a nucleic acid encoding a dominant negative RNase L or a dominant negative PKR, or both.

What is claimed is:  
     
         1 . A mutant mammalian cell having a homozygous disruption in the RNase L gene thereof and in the PKR gene thereof, wherein said mutant mammalian cell lacks biologically active RNase L enzyme and biologically active PKR enzyme.  
     
     
         2 . The mutant cell of  claim 1  wherein said cell is a mouse cell.  
     
     
         3 . A method for producing high levels of an exogenous, recombinant protein comprising: 
 a) providing a mutant mammalian cell having a homozygous disruption in its RNase L gene, or its PKR gene, or both, wherein said mutant mammalian cell lacks biologically active RNase L enzyme, or biologically active PKR enzyme, or both: 
 b) introducing a first nucleic acid encoding the exogenous protein into the mutant mammalian cell;  
 c) expressing said exogenous protein in said cell; and  
 d) isolating said exogenous protein from said cell.  
   
     
     
         4 . The method of  claim 3  wherein said mutant mammalian cell has a homozygous disruption in its RNase L gene and in its PKR gene, further comprising: 
 introducing a second nucleic acid encoding adenovirus VAI RNA or VA II RNA into said cell, and co-expressing said exogenous protein and said adenovirus RNA.  
 
     
     
         5 . The method of  claim 3 , wherein said mutant mammalian cell has a homozygous disruption in its RNase L gene, further comprising: 
 introducing a second nucleic acid encoding adenovirus VAI RNA or adenovirus VA II RNA into said cell, and co-expressing said exogenous protein and said adenovirus RNA.    
     
     
         6 . The method of  claim 3 , wherein said mutant mammalian cell has a homozygous disruption in its RNase L gene, further comprising: 
 introducing a second nucleic acid encoding a dominant negative PKR polypeptide into said cell, and co-expressing said exogenous protein and said dominant negative PKR polypeptide.    
     
     
         7 . The method of  claim 6  further comprising introducing a third nucleic acid encoding adenovirus VAI RNA or adenovirus VA II RNA into said cell, and co-expressing said exogenous protein, said dominant negative PKR polypeptide, and said adenovirus RNA.  
     
     
         8 . The method of  claim 3 , wherein said cell has a homozygous disruption in its PKR gene and lacks biologically active PKR enzyme.  
     
     
         9 . The method of  claim 8 , further comprising: introducing a second nucleic acid encoding adenovirus VAI RNA or adenovirus VA II RNA into said cell, and co-expressing said exogenous protein and said adenovirus RNA  
     
     
         10 . The method of  claim 8  further comprising: introducing a second nucleic acid encoding a dominant negative RNase L polypeptide into said cell, and co-expressing said exogenous protein and said dominant negative RNase L polypeptide.  
     
     
         11 . The method of  claim 10  further comprising: 
 introducing a third nucleic acid encoding adenovirus VAI RNA or adenovirus VA II RNA into said cell, and co-expressing said exogenous protein, said dominant negative RNase L polypeptide, and said adenovirus RNA  
 
     
     
         12 . A method for enhancing production of an exogenous, recombinant protein in a mammalian cell having an RNase L +/+ ; PKR +/+  genotype comprising: 
 a) introducing a first nucleic acid encoding the exogenous protein into said mammalian cell;  
 b) introducing a second nucleic acid encoding a dominant negative RNase L polypeptide or a dominant defective PKR polypeptide into said cell  
 c) co-expressing said exogenous protein and said dominant negative polypeptide in said cell; and  
 d) isolating said exogenous protein from said cell.  
 
     
     
         13 . The method of  claim 12  wherein said second nucleic acid encodes a dominant negative RNase L polypeptide.  
     
     
         14 . The method of  claim 12  wherein said second nucleic acid encodes a dominant negative PKR polypeptide.  
     
     
         15 . The method of  claim 13  further comprising: 
 introducing a third nucleic acid encoding adenovirus VA 1 RNA or Va II RNA into said cell and co-expressing said exogenous protein, said dominant negative RNase L polypeptide, and said adenovirus RNA in said cell.  
 
     
     
         16 . The method of  claim 14  further comprising 
 introducing a third nucleic acid encoding adenovirus VA 1 RNA or VA II RNA into said cell and co-expressing said exogenous protein, said dominant negative PKR polypeptide, and said adenovirus RNA in said cell.  
 
     
     
         17 . The method of  claim 13  further comprising: 
 introducing a third nucleic acid encoding a dominant negative PKR polypeptide and co-expressing said exogenous protein, said dominant negative RNase L polypeptide, and said dominant defective PKR polypeptide in said cell.  
 
     
     
         18 . The method of  claim 17  further comprising: 
 introducing a fourth nucleic acid encoding adenovirus VA I RNA or VA II RNA in said cell; and co-expressing said exogenous protein, said dominant negative RNase L polypeptide, said dominant negative PKR polypeptide, and said adenovirus RNA in said cell.  
 
     
     
         19 . A method for preparing mutant non-human mammalian cells for producing high levels of recombinant proteins, comprising: 
 a) intercrossing a first non-human mammal with a second non-human mammal of the same species, said first non-human mammal having a heterozygous disruption in its RNase L gene and said second non-human mammal having a heterozygous disruption in its PKR gene;    b) characterizing the embryos produced by step (a) to identify an embryo having an RNase L −/− : PKR −/−  genotype; and    c) isolating somatic cells from the embryo having said RNase L −/− : PKR −/−  genotype.    
     
     
         20 . The method of  claim 20  wherein said mammal is a mouse and wherein said somatic cells are fibroblasts.