Vector and method for achieving high level of expression in eukaryotic cells

A method of manufacturing an isolated animal cell transfected with a vector, said method comprising:

• (a) transfecting an animal cell with the vector, said vector comprising:
• (i) a selectable marker gene comprising a promoter operatively linked to a nucleic acid encoding a selectable marker enzyme which enables the animal cell to survive in a medium containing a compound normally toxic to the animal cell;
• (ii) a transcription unit comprising a promoter operatively linked to a nucleic acid encoding a protein;
• (iii) an enhancer located between the selectable marker gene and the transcription unit, which enhancer stimulates transcription of both the selectable marker gene and the transcription unit compared to the transcription of both the selectable marker gene and the transcription unit in the absence of the enhancer;
• and
• (iv) a blocking element comprising a promoter interposed between the enhancer and the selectable marker gene, which blocking element selectively attenuates the stimulation of transcription of the selectable marker gene;
• (b) culturing an animal cell resulting from step (a) in a medium containing the compound normally toxic to the animal cell;
• and
• (c) isolating an animal cell resulting from step (b) which expresses the protein.

An expression vector comprising:

• (a) a selectable marker gene comprising a promoter operatively linked to a nucleic acid encoding a selectable marker enzyme;
• (b) a transcription unit comprising a promoter operatively linked to a nucleic acid encoding a protein;
• (c) an enhancer located between the selectable marker gene and the transcription unit, which enhancer stimulates transcription of both the selectable marker gene and the transcription unit compared to the transcription of both the selectable marker gene and the transcription unit in the absence of the enhancer;
• and
• (d) a blocking element comprising a promoter interposed between the enhancer and the selectable marker gene, which blocking element selectively attenuates the stimulation of transcription of the selectable marker gene.