Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate

Abstract

This IB4-SAP-mediated method of selection of alphagal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement.

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

“…Targeted toxin technology using IB4SAP was first applied for specific elimination of porcine cells that were untransfected, or weakly expressed the EndoGalC gene, to create genetically modified cells suitable for pig-to-human xenotransplantation [8]. On the basis of this previous study, we decided to utilize this novel system for selecting transgene high-expressors, as described in Figure 1.…”

“…The Clostridium perfringens -derived endo-β-galactosidase C (EndoGalC) is known to digest the α-Gal epitope [6,7]. Therefore, introduction of an EndoGalC construct into the porcine genome has been considered as a promising approach to generate genetically modified piglets suitable for xenotransplantation [7,8,9]. In addition, the absence of an α-Gal epitope can be easily monitored by staining cells with Bandeiraea simplicifolia isolectin-B 4 (BS-I-B 4 , IB4), a lectin that specifically binds to the α-Gal epitope [1].…”

“…Therefore, targeted toxins have been considered as a powerful tool for removing unwanted cells from a pool of genetically modified population. In fact, we have previously demonstrated successful application of this technology for the isolation of transfectants with high transgene expression from among porcine embryonic fibroblasts (PEFs) transfected with the EndoGalC construct [8]. Moreover, the elimination of unwanted cells, including those that are untransfected and those weakly expressing the α-Gal epitope (considered as cells with low transgene expression), can be performed simply by incubating the target cells with SAP-conjugated IB4 (hereafter referred to as “IB4SAP”) for a short period, followed by culture under normal conditions.…”

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

“…In our previous experiments (Akasaka et al, 2010), we demonstrated that porcine embryonic fibroblasts (PEFs) expressing the EndoGalC gene strongly can survive after treatment with IB4 conjugated with saporin (hereafter referred to as IB4-SAP), but those that do so weakly or not at all died within 3 or 4 days after the treatment. When the surviving cells were inspected for possible expression of -Gal epitope on their cell surface using fluorescencelabeled IB4, no distinct fluorescence was noted, indicating the success of the targeted toxin technology.…”

“…In this context, targeted toxins are useful as a powerful tool for removing unwanted cells from a pool of GM cells. In fact, negative selection using targeted toxins has already been proven useful in vivo (Wiley and Kline, 2000;Vulchanova et al, 2001;Tarpley et al, 2004) and in vitro (Akasaka et al, 2010).…”