Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression
AbstractAlmost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cell surface. Almost all the surviving colonies after IB4SAP treatment were in fact negative for BS-I-B4 staining, and also strongly expressed EGFP. This system would be particularly valuable for researchers who wish to perform large-scale production of therapeutically important recombinant proteins.
Scifeed alert for new publicationsNever miss any articles matching your research from any publisher
- Get alerts for new papers matching your research
- Find out the new papers from selected authors
- Updated daily for 49'000+ journals and 6000+ publishers
- Define your Scifeed now
Sato, M.; Akasaka, E.; Saitoh, I.; Ohtsuka, M.; Nakamura, S.; Sakurai, T.; Watanabe, S. Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression. Biology 2013, 2, 341-355.
Sato M, Akasaka E, Saitoh I, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S. Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression. Biology. 2013; 2(1):341-355.Chicago/Turabian Style
Sato, Masahiro; Akasaka, Eri; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi. 2013. "Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression." Biology 2, no. 1: 341-355.