State Key Laboratory for AgroBiotechnology, China Agricultural University, Beijing, China.
Theriogenology. 2013 Jul 1;80(1):50-7. doi: 10.1016/j.theriogenology.2013.03.008. Epub 2013 Apr 25.
An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.
通过将 TLR4 插入到一个重建的 p3S-LoxP 质粒中，建立了一个高度表达 TLR4 的绵羊胎儿成纤维细胞系。通过将 TLR4 转染的胎儿成纤维细胞转移到中期（M）II 期去核卵母细胞（使用 SCNT）中，产生了过表达 TLR4 的转基因绵羊。由于使用延迟激活方法在体内成熟的来自 MII 期去核卵母细胞的重构胚胎的妊娠率（18.52%）高于在体外成熟的来自 MII 期去核卵母细胞的重构胚胎，因此使用前者程序。通过聚合酶链反应和 Southern blot 分析确认了 9 只 TLR4 转基因活产。通过逆转录聚合酶链反应和免疫组织化学分别验证了转基因羔羊耳部组织中 TLR4 在 mRNA 和蛋白质水平的表达增加。在脂多糖刺激后 0、1 和 4 小时，从 3 个月大的 TLR4 转基因而非非转基因羔羊中收集的外周血单核细胞和/或巨噬细胞中表达了更多的 toll 样受体 4 蛋白。此外，TLR4 转基因羔羊的单核细胞和/或巨噬细胞分泌的干扰素-γ 和肿瘤坏死因子-α在 1 小时时显著升高。因此，TLR4 转基因羔羊的单核细胞和/或巨噬细胞中的脂多糖诱导的炎症反应发生得更早，与增强的宿主免疫反应一致。总之，过表达 TLR4 的转基因绵羊是研究转基因动物在抗病性中的作用的主要模型，并且具有培育抗病性绵羊的潜力。
