Methods for making recombinant cells

A method for producing a cell line featuring expression of an amino acid sequence at high levels, the method comprising:

• a) introducing into host cells once or more than once a first vector comprising first selectable sequence operably linked to a segment encoding at least one copy of the amino acid sequence,
• b) culturing the host cells under conditions conductive to selecting the first vector,
• c) isolating cells expressing the amino acid sequence at a first expression level to produce a cell line (first high expressing cells), wherein the method further comprises:
• d) introducing into the first high expressing cells once or more than once a second vector encoding the amino acid sequence;
• and
• e) subjecting the cells to conditions conducive to expressing the amino acid sequence at a second expression level higher than the first expression level, and isolating cells expressing the amino acid sequence at the second expression level to produce a cell line (second high expressing cells).

A method for producing a cell line featuring increased expression of an amino acid sequence, the method comprising:

• a) introducing into host cells a first vector comprising a first selectable sequence operably linked to a segment encoding at least one copy of the amino acid sequence,
• b) culturing the host cells under conditions conducive to selecting the first vector,
• c) isolating cells expressing the amino acid sequence at a first expression level;
• and
• d) subjecting the cells to conditions conducive to expressing the amino acid sequence at a second expression level higher than the first expression level to produce the cell line,
• wherein the isolation further comprises selecting cells expressing a cell-surface marker.

A method for producing a genetically stable cell line featuring increased expression of an amino acid sequence, the method comprising:

• a) introducing into host cells a first vector comprising a first selectable sequence operably linked to a segment encoding at least one copy of the amino acid sequence,
• b) culturing the host cells under conditions conducive to selecting the first vector,
• c) isolating cells expressing the amino acid sequence at a first expression level,
• d) subjecting the cells to conditions conducive to expressing the amino acid sequence at a second expression level higher than the first expression level;
• and
• e) detecting the first vector within the genome of the cells to produce the genetically stable cell line,
• wherein the method further comprises selecting cells expressing a cell-surface marker.

A method for producing a multi-subunit protein, the method comprising:

• a) introducing into host cells a first vector comprising a first selectable sequence operably linked to a segment encoding at least one copy of a first subunit of the protein,
• b) culturing the host cells under conditions conducive to selecting the first vector,
• c) isolating cells expressing the amino acid sequence at a first expression level (first high expressing cells),
• d) introducing into the cells expressing at the first expression level a second vector encoding at least one copy of a second subunit of the protein,
• e) subjecting the cells to conditions conducive to expressing the amino acid sequence at a second expression level higher than the first expression level;
• and
• f) isolating the cells expressing the amino acid sequence at the second expression level (second high expressing cells) to produce the multi-subunit protein.