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Open AccessArticle

Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene Expression

1
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
2
Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan
3
Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan
4
Department of Surgery, National Defense Medical College, Saitama 359-8513, Japan
5
Department of Organ Regeneration, Graduate School of Medicine, Shinshu University, Nagano 390-8621, Japan
6
Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan
*
Author to whom correspondence should be addressed.
Biology 2013, 2(1), 341-355; https://doi.org/10.3390/biology2010341
Received: 24 December 2012 / Revised: 24 December 2012 / Accepted: 29 January 2013 / Published: 28 February 2013
(This article belongs to the Special Issue Gene Expression and Regulation)
Almost 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. View Full-Text
Keywords: a-Gal epitope; BS-I-B4 lectin; endo-b-galactosidase C; porcine embryonic fibroblasts; saporin; targeted toxin; high transgene expression a-Gal epitope; BS-I-B4 lectin; endo-b-galactosidase C; porcine embryonic fibroblasts; saporin; targeted toxin; high transgene expression
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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.

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