Toll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response. Using the technology of pronuclear microinjection, genetically modified (GM) sheep with TLR4 overexpression were generated. Previous studies have shown that these GM sheep exhibited a higher inflammatory response to Gram-negative bacteria infection than wild type (WT) sheep. In order to evaluate the gene expression of GM sheep and study the co-expressed and downstream genes for TLR4, peripheral blood mononuclear cells (PBMC) from TLR4-overexpressing (Tg) and wild type (WT) sheep were selected to discover the transcriptomic differences using RNA-Seq. An average of 18,754 and 19,530 known genes were identified in the Tg and WT libraries, respectively. A total of 338 known genes and 85 novel transcripts were found to be differentially expressed in the two libraries (p < 0.01). A differentially expressed genes (DEGs) enrichment analysis showed that the GO terms of inflammatory response, cell recognition, etc. were significantly (FDR < 0.05) enriched. Furthermore, the above DEGs were significantly (FDR < 0.05) enriched in the sole KEGG pathway of the Phagosome. Real-time PCR showed the OLR1, TLR4 and CD14 genes to be differentially expressed in the two groups, which validated the DEGs data. The RNA-Seq results revealed that the overexpressed TLR4 in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in PBMC.

中文翻译：

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评估过表达 TLR4 对绵羊外周血单个核细胞转录组谱的影响。

Toll 样受体 4 (TLR4) 在消除革兰氏阴性菌感染和启动抗炎反应中起重要作用。使用原核显微注射技术，产生了具有 TLR4 过表达的转基因 (GM) 羊。以前的研究表明，这些转基因绵羊对革兰氏阴性菌感染的炎症反应比野生型 (WT) 绵羊高。为了评估转基因绵羊的基因表达并研究 TLR4 的共表达和下游基因，选择来自 TLR4 过表达 (Tg) 和野生型 (WT) 绵羊的外周血单个核细胞 (PBMC) 来发现转录组差异使用 RNA-Seq。在 Tg 和 WT 文库中分别鉴定出平均 18,754 和 19,530 个已知基因。共有 338 个已知基因和 85 个新转录本被发现在两个文库中差异表达（p < 0.01）。差异表达基因(DEGs)富集分析表明炎症反应、细胞识别等GO术语显着富集(FDR < 0.05)。此外，上述DEG在吞噬体的唯一KEGG途径中显着富集（FDR <0.05）。实时荧光定量PCR显示OLR1、TLR4和CD14基因在两组中均有差异表达，验证了DEGs数据。RNA-Seq 结果表明，我们实验中过表达的 TLR4 通过增加 PBMC 的吞噬作用来增强羊的先天免疫反应。差异表达基因(DEGs)富集分析表明炎症反应、细胞识别等GO术语显着富集(FDR < 0.05)。此外，上述DEG在吞噬体的唯一KEGG途径中显着富集（FDR <0.05）。实时荧光定量PCR显示OLR1、TLR4和CD14基因在两组中均有差异表达，验证了DEGs数据。RNA-Seq 结果表明，我们实验中过表达的 TLR4 通过增加 PBMC 的吞噬作用来增强羊的先天免疫反应。差异表达基因(DEGs)富集分析表明炎症反应、细胞识别等GO术语显着富集(FDR < 0.05)。此外，上述DEG在吞噬体的唯一KEGG途径中显着富集（FDR <0.05）。实时荧光定量PCR显示OLR1、TLR4和CD14基因在两组中均有差异表达，验证了DEGs数据。RNA-Seq 结果表明，我们实验中过表达的 TLR4 通过增加 PBMC 的吞噬作用来增强羊的先天免疫反应。TLR4和CD14基因在两组中均有差异表达，验证了DEGs数据。RNA-Seq 结果表明，我们实验中过表达的 TLR4 通过增加 PBMC 的吞噬作用来增强羊的先天免疫反应。TLR4和CD14基因在两组中均有差异表达，验证了DEGs数据。RNA-Seq 结果表明，我们实验中过表达的 TLR4 通过增加 PBMC 的吞噬作用来增强羊的先天免疫反应。