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

Biology

Masahiro Sato¹  Eri Akasaka²  Issei Saitoh²  Masato Ohtsuka³  Shingo Nakamura⁴  Takayuki Sakurai⁵ 

[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] E-Mails:

[4]Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan

[5] E-Mail:

[6]Department of Surgery, National Defense Medical College, Saitama 359-8513, Japan

[7] E-Mail:

[8]Department of Organ Regeneration, Graduate School of Medicine, Shinshu University, Nagano 390-8621, Japan

[9] E-Mail:

关键词: α-Gal epitope;    BS-I-B₄ lectin;    endo-β-galactosidase C;    porcine embryonic fibroblasts;    saporin;    targeted toxin;    high transgene expression;   

DOI  :  10.3390/biology2010341

来源: mdpi

【 摘 要 】

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-β-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface α-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (α-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 α-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.

【 授权许可】

CC BY   
© 2013 by the authors; licensee MDPI, Basel, Switzerland.