Selection of High Expressing Mammalian Cells by Surface Display of Reporters

Part of the Methods in Molecular Biology book series (MIMB, volume 801)


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 words

Flow cytometry Clone screen Fluorescent reporter Internal ribosome entry site 


  1. 1.
    Gurtu, V.; Yan, G.; Zhang, G., IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines. Biochem. Biophys. Res. Comm. 1996, 229, 295–298.PubMedCrossRefGoogle Scholar
  2. 2.
    Mizuguchi, H.; Xu, Z.; Ishii-Watabe, A.; Uchida, E.; Hayakawa, T., IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Molecular Therapy 2000, 1, 376–382.PubMedCrossRefGoogle Scholar
  3. 3.
    Liu, X.; Constantinescu, S.N.; Sun, Y.; Bogan, J.S.; Hirsch, D.; Weinberg, R.A.; Lodish, H.F., Generation of mammalian cells stably expressing multiple genes at predetermined levels. Anal. Biochem. 2000, 280, 20–28.PubMedCrossRefGoogle Scholar
  4. 4.
    Barnes, L.M.; Bentley, C.M.; Dickson, A.J., Stability of protein production from recombinant mammalian cells. Biotechnol. Bioeng. 2003, 81, 631–639.PubMedCrossRefGoogle Scholar
  5. 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
  6. 6.
    DeMaria, C.T.; Cairns, V.; Schwarz, C.; Zhang, J.; Guerin, M.; Zuena, E.; Estes, S.; Karey, K.P., Accelerated Clone Selection for Recombinant CHO Cells Using a FACS-Based High-Throughput Screen. Biotechnol. Prog. 2007, 23, 465–472.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Therapeutic Protein ExpressionGenzyme CorporationFraminghamUSA

Personalised recommendations