Production of recombinant proteins using herpes virus promoters and VP16 transactivators
AssigneeG. D. Searle & Co.
InventorsPaul Jerome Hippenmeyer, Maureen Katherine Highkin
Life of this patent
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Abstract
Stable cell lines are produced to express high levels of a gene product of interest using VP16, a herpes simplex virus transactivator, and a promoter from herpes simplex virus which is a target for VP16. The transactivator and promoter are introduced to a cell line separately using antibiotic resistance genes as selectable markers on separate vectors.
Claims20
A method of producing a mammalian cell line for stable expression of a gene product, comprising the steps of:
- cotransfecting a mammalian cell with a first construct which cause said cell to express herpes simplex virus transactivating protein VP16 and a second construct comprising a selectable resistance gene to a first antibiotic;
- selecting said cell which is resistant to the first antibiotic;
- screening said cell which is resistant to the first antibiotic for VP16 expression;
- cotransfecting said cell which expresses herpes simplex virus transactivating protein VP16 with third and fourth constructs, the third construct comprising a herpes simplex virus gene promoter that responds to VP16 operably linked to a gene of interest, and the fourth construct comprising a selectable resistance gene to a second antibiotic;
- selecting said cell which is resistant to the second antibiotic;
- screening said cell which is resistant to the second antibiotic for expression of the gene product of said gene of interest;
- and
- culturing said cell into a stable cell line.
A stable mammalian cell line which expresses a gene product, said cell line made by the process of:
- cotransfecting a mammalian cell with a first construct which cause said cell to express herpes simplex virus transactivating protein VP16 and a second construct comprising a selectable resistance gene to a first antibiotic;
- selecting said cell which is resistant to the first antibiotic;
- screening said cell which is resistant to the first antibiotic for VP16 expression;
- cotransfecting said cell which expresses herpes simplex virus transactivating protein VP16 with third and fourth constructs, the third construct comprising a herpes simplex virus gene promoter that responds to VP16 operably linked to a gene of interest, and the fourth construct comprising a selectable resistance gene to a second antibiotic;
- selecting said cell which is resistant to the second antibiotic;
- and
- screening said cell which is resistant to the second antibiotic for expression of the gene product of said gene of interest.
A cell line which stably expresses a gene product, said cell line comprising:
- a first construct which causes the cell to express herpes simplex virus transactivator protein VP16;
- a second construct comprising a herpes simplex virus gene promoter that responds to VP16 operably linked to a gene of interest;
- and
- a third and fourth construct each comprising a selectable resistance gene to a selectable agent.
Dependencies are inferred from the claim text.