Targeted Toxin as a Useful Reagent for Enrichment of a-Gal Epitope-Negative Cells Used for Somatic Cell Nuclear Transfer in Pigs

Chi, Haiying; Miyoshi, Katsuhiko

doi:10.5772/28364

Cited by 1 publication

(1 citation statement)

References 24 publications

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“…Furthermore, this system appears to be useful in the xenotransplantation field. We have recently succeeded in isolating porcine cells with greatly reduced expression of the α-Gal epitope after transfection with a vector expressing small interfering RNA (siRNA) for α-GalT [ 18 ]. We showed that when these isolated siRNA-expressing cells were used as donor cells for somatic cell nuclear transfer (SCNT) experiments in pigs, the resulting cloned blastocysts exhibited a significant reduction in α-Gal epitope expression.…”

Section: Resultsmentioning

confidence: 99%

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

Akasaka

Saitoh

Ohtsuka

et al. 2013

Biology

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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.

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Section: Resultsmentioning

confidence: 99%