Targeted toxin-based selectable drug-free enrichment of Mammalian cells with high transgene expression.
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.
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan. masasato@ms.kagoshima-u.ac.jp.
Masahiro
M
Sato
Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan. stylistics777@yahoo.co.jp.
Eri
E
Akasaka
Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan. isaito@dent.niigata-u.ac.jp.
Issei
I
Saitoh
Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
Masato
M
Ohtsuka
Department of Surgery, National Defense Medical College, Saitama 359-8513, Japan. snaka@ndmc.ac.jp.
Shingo
S
Nakamura
Department of Organ Regeneration, Graduate School of Medicine, Shinshu University, Nagano 390-8621, Japan. tsakurai@shinshu-u.ac.jp.
Takayuki
T
Sakurai
Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan. kettle@affrc.go.jp.
Satoshi
S
Watanabe