Generation of human beta Fucci cell lines.
Two different EndoC-βH2 Fucci derivatives were generated. (A) In one EndoC-βH2 Fucci cell line, the two genes encoding the Fucci indicators (GF: green Fucci, mAG-ΔGeminin and OF: Orange Fucci, mKO2-ΔCdt1) were stably transferred by sequential retroviral transduction using two bicistronic vectors. They encode either GF plus the gene encoding the resistance to zeocin (ZeoR), or OF plus the gene encoding the resistance to puromycin (PuroR). After each step, a selection using the appropriate drug was achieved and the resulting cells line, termed EndoC-βH2-OFP-GFZ, was routinely cultured in presence of both selective drugs. (B) In the other EndoC-βH2 Fucci cell line, the two genes encoding the Fucci indicators (GF: green Fucci, eGFP-ΔGeminin and OF: Orange Fucci, mKO2-ΔCdt1) were present on the same retrovector and separated by a 2A peptide together with the gene encoding the resistance to puromycin. They were thus simultaneously transferred and the resulting cell line, termed EndoC-βH2-PGF2AOF, was selected and routinely cultured in a puromycin-containing medium.