A GFP-based method facilitates clonal selection of transfected CHO cells

Denise Freimark (1) , Valérie Jérôme (1) , Ruth Freitag (2)

1 Chair for Process Biotechnology
2 Universität Bayreuth

The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows for clonal selection in standard 96-well cell culture plates by a simple fluorescence reading. The procedure combines the efficiency of green fluorescent protein/fluorescence-based screening in a plate reader with the limited dilution technique to select high-producing subclones. In particular, the genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site (IRES). In consequence, they are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. The method is an alternative to the identification of high-producer clones using various cell sorting methods, which often require dedicated equipment not necessarily available in all laboratories. Two examples are presented to demonstrate the robustness and performance of this technique, namely expression of the recombinant growth factors hIGF1A and hVEGFA in CHO cells.

Mots clés: Life Sciences

Denise Freimark, Valérie Jérôme, Ruth Freitag. A GFP-based method facilitates clonal selection of transfected CHO cells. Biotechnology Journal, 2009, 4 (10), pp.24. ⟨10.1002/biot.200800264⟩. ⟨hal-00514309⟩