Inventors:
Robert Silverman - Beachwood OH, US
Bryan Williams - Cleveland OH, US
Fulvia Terenzi - Cleveland OH, US
Aimin Zhou - Solon OH, US
Sandy Der - Toronto, CA
International Classification:
C12P021/02
C12N009/12
C12N005/06
US Classification:
435/069100, 435/194000, 435/354000, 435/320100
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.
