Evaluating the effect of TLR4-overexpressing on the transcriptome profile in ovine peripheral blood mononuclear cells

Abstract

Background: 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. Result: 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. Conclusions: The RNA-Seq results revealed that the overexpressed TLR4 in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in PBMC.

Transcriptomic meta-analysis reveals unannotated long non-coding RNAs related to the immune response in sheep

“…These genes were part of the iFMDV-associated co-expression module and many were also part of the DCN. The IFN response was mostly driven by the inactivated vaccine ( Braun et al, 2018 ; Jouneau et al, 2020 ) and the LPS stimulation datasets ( Bush et al, 2020 ), while the helminth infection ( Fu et al, 2016 ; Niedziela et al, 2021 ) and other smaller datasets ( Varela-Martínez et al, 2018 ; Wang et al, 2019 ; Guo et al, 2020 ) produced a different expression profile, as seen in the stimulation-correlated co-expression modules. In the same manner as known ISGs, lncRNAs can also be induced by IFN and have important roles in controlling pathogen infection and resolution of the immune response, or they can regulate the IFN mediated host defence ( Meng et al, 2017 ; Qiu et al, 2018 ).…”

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune‐related signaling pathways and gut microbiota

“…Subsequently, the library was used for sequencing on an Illumina Hiseq 2000 platform (Illumina, San Diego, CA, USA). Reads filtering, differential expression genes (DEGs) analysis, and enrichment analysis of GO/KEGG were performed according to Guo et al 30 DEGs of TLRs and NLRs families selected from the RNA-seq data were validated using qPCR, which was performed with LightCycler ® 480 SYBR Green I Master (Roche Diagnostics) on the LightCycler 480II System (Roche, Switzerland). The qPCR program included a 10 minutes incubation at 95℃ to activate FastStart DNA polymerase, followed by 35 cycles of 95℃ for 10 seconds, 60℃ for 15 seconds, and 72℃ for the appropriate extension time with single fluorescence acquisition.…”