College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu 210095, China.
Antiviral Res. 2013 Aug;99(2):158-64. doi: 10.1016/j.antiviral.2013.05.010. Epub 2013 Jun 2.
The purpose of this study was to enhance the production of transgenic cloned embryos with porcine reproductive and respiratory syndrome virus (PRRSV) shRNA expression cassettes. To construct transgenic vector with expression targeting against PRRSV, PRRSV shRNA expression cassettes were inserted into pEGFP-N1 and the ability of resulting recombinant plasmid pEGFP-G1 inhibiting virus replication was examined in Marc-145 cells. Results showed that PRRSV replication could be significantly inhibited by pEGFP-G1 in Marc-145 cells compared with the control. The pEGFP-G1 plasmid was used to deliver a transgene expressing EGFP and the PRRSV shRNA into porcine fetal fibroblasts (PFF). Fluorescent-positive cells were used as nuclear donors for somatic cell nuclear transfer (SCNT) to produce shRNA-EGFP transgenic cloned embryos. The effects of trichostatin A (TSA) on production of transgenic cloned embryos were investigated. Reconstructed embryos were designed into 4 groups: Donor cells of Group A were treated with 50nM TSA for 24h before SCNT. Reconstructed embryos of Group B were treated with 50nM TSA for 24h after activation. Both donor cells and reconstructed embryos in Group C were treated with TSA and Group D were the control without TSA treatment. The results showed no difference (p>0.05) in cleavage rates among the 4 groups; however, blastocyst developmental rates of Group B and C (30.9% and 42.0%, respectively) were higher than for Group A and D (21.2% and 22.1%, respectively) with Group C highest among groups (p<0.05). Interestingly, EGFP expression intensity of transgenic cloned blastocysts of Group A was the highest. Our results provide promising evidence toward a new approach for production of transgenic cloned pigs with resistance to PRRSV and possibly a wide variety of other porcine diseases.
本研究旨在提高带有猪繁殖与呼吸综合征病毒（PRRSV）shRNA 表达盒的转基因克隆胚胎的生产效率。为了构建靶向 PRRSV 的转基因载体，将 PRRSV shRNA 表达盒插入 pEGFP-N1 中，并在 Marc-145 细胞中检测由此产生的重组质粒 pEGFP-G1 抑制病毒复制的能力。结果表明，与对照组相比，pEGFP-G1 可显著抑制 Marc-145 细胞中的 PRRSV 复制。将 pEGFP-G1 质粒用于将表达 EGFP 和 PRRSV shRNA 的转基因递送到猪胎儿成纤维细胞（PFF）中。荧光阳性细胞被用作体细胞核移植（SCNT）的核供体，以生产 shRNA-EGFP 转基因克隆胚胎。研究了曲古抑菌素 A（TSA）对生产转基因克隆胚胎的影响。将重构胚胎设计为 4 组：A 组的供体细胞在 SCNT 前用 50nM TSA 处理 24 小时。B 组的重构胚胎在激活后用 50nM TSA 处理 24 小时。C 组的供体细胞和重构胚胎均用 TSA 处理，D 组为无 TSA 处理的对照组。结果表明，4 组的卵裂率无差异（p>0.05）；然而，B 组和 C 组的囊胚发育率（分别为 30.9%和 42.0%）高于 A 组和 D 组（分别为 21.2%和 22.1%），其中 C 组最高（p<0.05）。有趣的是，A 组转基因克隆囊胚的 EGFP 表达强度最高。我们的结果为生产具有抗 PRRSV 能力的转基因克隆猪提供了有希望的证据，并且可能为其他多种猪病提供了广泛的应用前景。
