A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells
A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells
复制标题
DOI：
10.1002/biot.201400283
复制
发表时间：
2015-01-01
影响因子：
4.7
通讯作者：
Watanabe, Satoshi
中科院分区：
文献类型：
--
作者：
Sato, Masahiro;Inada, Emi;Watanabe, Satoshi
携带外源DNA的细胞的分离通常通过引入质粒来实现，但其效率仍然很低。在这项研究中，我们开发了一种新的策略，以获得稳定的转染有效。用两种质粒转染猪胚胎成纤维细胞：（i）pTransIEnd，其包含普遍存在的启动子、piggyBac（PB）转座酶基因、内部核糖体进入位点、产气荚膜梭菌来源的内切β-半乳糖苷酶C（ii）基于PB的质粒，称为pT-EGFP，其含有增强型绿色荧光蛋白（EGFP）表达单位，其侧翼为PB受体位点。PB转座酶可以加速转座子载体的染色体整合。EndoGalC表达导致细胞表面Gal表位的去除，其被Bandeiraea simplicifolia异凝集素-B-4（IB 4）特异性识别。转染后4天，将细胞用IB 4SAP（与皂草素缀合的IB 4，其消除了任何-Gal表位表达细胞）处理短时间，然后标准培养约10天。出现了几个菌落，其中大多数是EGFP表达阳性，缺乏transIEend。这些结果表明，所提出的方法是有用的和有效的获得稳定的转染不使用耐药基因，并提供了一种新的途径，在培养的非人类哺乳动物细胞的基因操作。
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--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 -Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B-4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any -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.