Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells

Mammalian cells are often used for the expression of recombinant proteins. The process of screening transfected cells randomly for high producing clon es is tedious and time consuming. We evaluated using green fluorescent prot ein (GFP) for selection of high producing clones by fluorescence-activated cell sorter (FACS) to reduce screening effort. We expressed neurotrophin-3 (NT3), deoxyribonuclease (DNase), or vascular endothelial growth factor (VE GF) with GFP in Chinese hamster ovary cells. The Vector expressed the desir ed secreted protein and the selectable marker, dihydrofolate reductase, in one expression unit and the intracellular GFP in a second expression unit. Transfected cells were grown in selection medium and sorted by FACS. High f luorescence clones were obtained and found to produce high amounts of the d esired protein; VEGF productivity correlated well with GFP fluorescence in 48 clones. Further studies demonstrated that productivity correlated very w ell with RNA of the desired protein. For comparison, we randomly picked and screened 144 VEGF clones, and the highest producing VEGF clone obtained pr oduced 0.7 pg/cell/day. In contrast, the highest producing VEGF clone obtai ned by FACS sorting produced 4.4 pg/cell/day. FACS sorting therefore select ed high producing clones efficiently. Since an assay for the desired protei n is not required, high producing clones for a protein of unknown function can be obtained by FACS sorting followed by measuring the RNA level of the desired protein in the highly fluorescent clones. (C) 2000 Elsevier Science B.V. All rights reserved.