Guo Xiaofei, Zhang Jinlong, Li Yao, Yang Jing, Li Yihai, Dong Chunxiao, Liu Guoshi, Lian Zhengxing, Zhang Xiaosheng
Tianjin Institute of Animal Husbandry and Veterinary Medicine, Tianjin Academy of Agricultural Sciences, Tianjin, 300381 China.
College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
J Biol Res (Thessalon). 2020 Jul 29;27:13. doi: 10.1186/s40709-020-00124-3. eCollection 2020 Dec.
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 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 , peripheral blood mononuclear cells (PBMC) from -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 ( < 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 , and genes to be differentially expressed in the two groups, which validated the DEGs data.
The RNA-Seq results revealed that the overexpressed in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in PBMC.
Toll样受体4（TLR4）在清除革兰氏阴性菌感染及启动抗炎反应中发挥重要作用。利用原核显微注射技术，培育出了过表达的转基因（GM）绵羊。先前的研究表明，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）绵羊更强。为了评估转基因绵羊的基因表达情况并研究TLR4的共表达基因和下游基因，我们选取了过表达（Tg）绵羊和野生型（WT）绵羊的外周血单个核细胞（PBMC），采用RNA测序技术来发现转录组差异。
在Tg文库和WT文库中分别平均鉴定出18,754个和19,530个已知基因。共发现338个已知基因和85个新转录本在两个文库中差异表达（P < 0.01）。差异表达基因（DEG）富集分析表明，炎症反应、细胞识别等GO术语显著富集（FDR < 0.05）。此外，上述DEG在吞噬体的唯一KEGG途径中显著富集（FDR < 0.05）。实时定量PCR显示两组中TLR4、MyD88和NF-κB基因差异表达，验证了DEG数据。
RNA测序结果表明，我们实验中过表达的TLR4通过增强PBMC中的吞噬作用来强化绵羊的固有免疫反应。
