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

Abstract

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.

Generation of α‐1,3‐Galactosyltransferase‐Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in of a Small Mutated Sequence and a Targeted Toxin‐Based Selection System

“…Next, the remaining portion of the trypsinized cells (10 5 cells) was subjected to short treatment (2 h, 37°C) with IB4SAP to enrich for a-gal epitope-negative cells that might have been successfully knocked-in. This treatment eliminates a-gal epitope-expressing cells, such as untransfected cells and transfected cells that still express the a-gal epitope (Akasaka et al 2010;Sato et al 2013). After treatment, the remaining cells were cultured in normal medium.…”

“…Because the efficiency of the CRISPR/Cas9-mediated knock-in system is generally low (Harrison et al 2014;Hsu et al 2014), we employed a targeted toxin-based drug-free selection system using a commercially available saporin (SAP)-conjugated IB4 (IB4SAP). This enables the enrichment of a-gal epitope-negative stable transfectants after a short incubation of transfected porcine cells with the IB4SAP-containing solution and subsequent cultivation in normal medium (Akasaka et al 2010;Sato et al 2013).…”

A combination of targeted toxin technology and the piggyBac‐mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells

“…pT‐pac carries a puromycin resistance (puromycin acetyltransferase) gene ( pac ) expression unit (CAG promoter + pac + poly[A] sites). pTransIEnd was constructed by substituting the Eco RI/ Bgl II 1.9‐kb fragment containing a transposase gene with EGFP cDNA in pCEIEnd plasmid [1], which is comprised of CAG, a 0.9‐kb EGFP cDNA, a 0.63‐kb internal ribosomal entry site (IRES), and the 3‐kb EndoGalC gene encoding for a cytoplasmic tail and a transmembrane domain; the stem region of the pig α‐GalT cDNA [7]. Therefore, when the EndoGalC protein is expressed in cells, it is retained at the cell membrane where it then exerts enzymatic activity.…”

“…However, the toxic and deleterious effects of such drugs often cause dysfunction in normal cells. Thus, drug‐free selection of recombinants would be a preferred approach; however, few reports on this approach have been available to date [1, 2].…”

“…The surviving cells are then those that do not express the α‐Gal epitope on their cell surface. Based on these findings, we have proven that coexpression of a gene of interest (GOI) and EndoGalC, along with subsequent IB4SAP treatment, can be used to enrich α‐Gal epitope‐negative cells that strongly express the GOI [1].…”