A GFP-based method facilitates clonal selection of transfected CHO cells.
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 clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus 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. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.
Freimark
Denise
D
Chair for Process Biotechnology University of Bayreuth, Germany.
Jèrôme
Valèrie
V
Freitag
Ruth
R
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D019976:Cloning, Organism / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection / Q000379:methods*
D049452:Green Fluorescent Proteins