High-throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein-GFP fusion protein.
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 microg hGH/10(6) 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.
Bailey
Charles G
CG
Department of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, Australia.
Tait
A Sasha
AS
Sunstrom
Noelle-Ann
NA
D000818:Animals; D016466:CHO Cells / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008668:Metallothionein / Q000032:analysis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012641:Selection, Genetic; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013223:Statistics as Topic; D014162:Transfection / Q000379:methods
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008668:Metallothionein