Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE).
We have recently reported on the isolation of a 5.7 kb segment of Chinese hamster ovary cell genomic DNA, Expression Augmenting Sequence Element (EASE), which when used in bicistronic expression vectors allows the development of stable Chinese hamster ovary cell pools in a five to seven week time period that express high levels of recombinant protein (6-25 mug 10-6 cells/day depending on the protein). In the present study, we have mapped the activity of the EASE to a 2.1 kb region using colony forming assays and developed bicistronic expression vectors with the smaller EASE or control lambda DNA. The recovery of pools expressing the hematopoietic growth factor, FLT3 Ligand, in methotrexate-containing media took 1 to 4 weeks less when using EASE expression vectors compared with control vectors. The cell pools developed with the EASE and control vectors had similar final protein expression levels. Southern blot analysis suggested the expression cassette from the EASE containing vectors integrated in tandem arrays arranged in either head to head or head to tail fashion. By contrast, control vectors appeared to integrate with multiple interruptions to the expression vector. Thus, the EASE, within a bicistronic expression vector, appeared to facilitate tandem vector integration and reduce the time required to develop cell pools for protein expression.
Aldrich
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Immunex Corporation, 51 University Street, Seattle, Washington, 98101, U.S.A.
Thomas
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Morris
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