Host Cells Containing Multiple Integrating Vectors
AssigneeOrganization Name Catalent Pharma Solutions
InventorsRobert D. Bremmel, Linda U. Miller, Gregory T. Bleck
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Abstract
The present invention relates to the production of proteins in host cells, and more particularly to host cells containing multiple integrated copies of an integrating vector. Suitable integrating vectors for use in the present invention include retrovirus vectors, lentivirus vectors, transposon vectors, and adeno-associated virus vectors. Methods are provided in which the host cells are prepared by using the integrating vectors at a high multiplicity of infection. The host cells are useful for producing pharmaceutical proteins, variants of proteins for use in screening assays, and for direct use in high throughput screening.
Claims102
A host cell comprising a genome, said genome comprising at least two integrated integrating vectors, wherein said integrating vectors comprise at least one exogenous gene operably linked to a promoter.
A method for transfecting host cells comprising:
- 1) providing:
- a) a host cell comprising a genome, and
- b) a plurality of integrating vectors;
- and
- 2) contacting said host cell with said plurality of integrating vectors under conditions such that at least two integrating vectors integrate into said genome of said host cell.
A method of producing a protein of interest comprising:
- 1) providing a host cell comprising a genome, said genome comprising at least two integrated copies of at least one integrating vector comprising an exogenous gene operably linked to a promoter, wherein said exogenous gene encodes a protein of interest, and
- 2) culturing said host cells under conditions such that said protein of interest is produced.
A method for screening compounds comprising:
- 1) providing:
- a) providing a host cell comprising a genome, said genome comprising at least two integrated copies of at least one integrating vector comprising an exogenous gene operably linked to a promotor, wherein said exogenous gene encodes a protein of interest;
- and
- b) one or more test compounds;
- 2) culturing said host cells under conditions such that said protein of interest is expressed;
- 3) treating said host cells with said one or more test compounds;
- and
- 4) assaying for the presence of a response in said host cells to said test compound.
A method for comparing protein function comprising:
- 1) providing
- a) a first host cell comprising a first integrating vector comprising a promoter operably linked to a first exogenous gene, wherein said first exogenous gene encodes a first protein of interest;
- b) at least a second host cell comprising a second integrating vector comprising a promoter operably linked to a second exogenous gene, wherein said second exogenous gene encodes a second protein of interest that is a variant of said first protein of interest;
- 2) culturing said host cells under conditions such that said first and second proteins of interest are produced;
- and
- 3) comparing the activities of said first and second proteins of interest.
A method comprising:
- 1) providing:
- a) a host cell comprising a genome comprising at least one integrated exogenous gene;
- and
- b) a plurality of integrating vectors;
- and
- 2) contacting said host cell with said plurality of integrating vectors under conditions such that at least two of said integrating vectors integrate into said genome of said host cell.
Dependencies are inferred from the claim text.