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

Abstract

Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells.

piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential

“…The PB-based transposons and PB expression plasmid used in the present study are shown in Figure 1A. Briefly, the pTrans (pCX-mPB) vector [37] confers expression of the PB transposase gene under the control of CAG , a chicken β-actin-based systemic promoter [38]. pT-pac is a transposon vector carrying a puromycin acetyltransferase gene ( pac ) expression unit linked to the mouse phosphoglycerate kinase ( Pgk ) promoter [37].…”

The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones

“…Thus, cells surviving after IB4SAP treatment must be those exhibiting strong expression of EndoGalC (as well as CRISPR/Cas9-related components), although its expression was transient. Notably, Sato et al [ 23 , 24 ] demonstrated that high expression levels of EndoGalC (evaluated by the loss of the α-Gal epitope) are closely associated with those of GFP from the co-transfected expression vector. From these present studies, our targeted toxin-based cell selection system was found to be more effective for the enrichment of bi-allelic KO clones than the conventional puromycin-based cell selection system.…”

“…Therefore, cells transfected with an EndoGalC expression vector fail to react with IB4 [ 22 ]. Using this unique inverse-relationship between decreased reactivity to IB4 and increased EndoGalC expression, Sato et al [ 23 , 24 ] developed a novel drug-free selection system for genetically modified cells. Based on the background of this study, we developed a novel CRISPR/Cas9-based approach to enrich genome-edited non-human cells having bi-allelic KO alleles in a drug-free condition.…”

Intravenous Delivery of piggyBac Transposons as a Useful Tool for Liver-Specific Gene-Switching

“…Two PB expression plasmids pT-EGFP ( Figure 1 A) and pT-CETD ( Figure 2 A) were generated using pPB (pPB-MCS-P5), a plasmid carrying two PB acceptors with inverted repeats, as a basal plasmid. pT-EGFP carries an EGFP gene expression unit (CAG + EGFP cDNA + poly(A) sites) [ 62 ]. pT-CETD confers gene switching from EGFP cDNA to the DT-A gene when Cre expression occurs, as previously discussed [ 37 ].…”