SCREENING AND ENRICHMENT SYSTEM FOR PROTEIN EXPRESSION IN EUKARYOTIC CELLS USING A TRICISTRONIC EXPRESSION CASSETTE
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United States of America Patent
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Mar 13, 2014
app pub date -
Aug 20, 2013
filing date -
Aug 21, 2012
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Abandoned
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Abstract
The present invention provides a polynucleotide and a method for screening and enriching a recombinant protein expressed in a eukaryotic cell, the polynucleotide comprising a tricistronic expression cassette comprising a) a promoter, b) a gene of interest (GOI), c) a reporter gene, d) a selection marker gene, e) an internal ribosome entry site (IRES) element, and f) a 2A element. Said method comprises a) transfecting or transducing suitable eukaryotic host cells with the polynucleotides of the present invention, b) culturing the eukaryotic host cells under conditions so as to express the protein of interest, the reporter protein and the selection marker protein in a cell culture selection medium suitable for selecting positively-transfected/transduced cells by means of said selection marker, and c) screening, sorting and/or enriching the cells expressing high levels of protein of interest by means of the reporter protein.
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