Selection of High Expressing Mammalian Cells by Surface Display of Reporters
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A flow cytometry method using a nonfluorescent reporter protein was developed for rapid, early-stage identification of cells producing high levels of a recombinant protein of interest. A cell surface reporter protein is coexpressed with the protein of interest, and the reporter protein is detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the protein of interest are linked by an IRES so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein’s expression level accurately predicts, on a per cell basis, the relative expression level of the protein of interest. This method provides an effective process for selecting cells that express high levels of recombinant proteins, with the benefits of rapid and accurate 96-well plate clone screening (that is both quantitative and qualitative) and elimination of unstable clones during subsequent scale up and culture. Furthermore, because this method does not rely on the availability of a detection reagent specific for the protein of interest that is expressed, it can be easily implemented into any cell line development process.
Key wordsFlow cytometry Clone screen Fluorescent reporter Internal ribosome entry site
- 5.Davies, M.V. and Kaufman, R.J.; The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation. J.Virol. 1992, 66, 1924–1932.Google Scholar