High‐throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein‐GFP fusion protein

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Abstract
Transfected mammalian cells can be used for the production of fully processed recombinant proteins for medical and industrial purposes. However, the isolation of high‐producing clones is traditionally time‐consuming. Therefore, we developed a high‐throughput screening method to reduce the time and effort required to isolate high‐producing cells. This involved the construction of an expression vector containing the amplifiable gene metallothionein (MT), fused in‐frame to green fluorescent protein (GFP). The fusion gene (MTGFP) confers metal resistance similar to that of the wild‐type metallothionein and expression can be monitored using either flow cytometry or a fluorometer to measure green fluorescence. Expression of MTGFP acted as a dominant selectable marker allowing rapid and more efficient selection of clones at defined metal concentrations than with the antibiotic G418. Cells harboring MTGFP responded to increasing metal concentrations with a corresponding increase in fluorescence. There was also a corresponding increase in recombinant protein production, indicating that MTGFP could be used as a selectable and amplifiable gene for the coexpression of foreign genes. Using our expression vector encoding MTGFP, we demonstrate a high‐throughput clonal selection protocol for the rapid isolation of high‐producing clones from transfected CHO cells. We were able to isolate cell lines reaching specific productivities of >10 μg hGH/106 cells/day within 4 weeks of transfection. The advantage of this method is that it can be easily adapted for automated procedures using robotic handling systems. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 670–676, 2002.