Fig. 1

Detection of TLR4 overexpression in ovine blood monocytes. a Schematic diagram of the structure of the CMV-Ovis aries TLR4 overexpression vector. b Representative Southern blot used to identify transgenic (TG) sheep based on the presence of the TLR4 transgene. Partially TG sheep had both the endogenous 4700-bp TLR4 band and the exogenous 2771-bp TLR4 band. Marker, 1-kb ladder; lanes 1–8, eight individuals comprising the wild-type (WT) sheep in lanes 1 and 3 and the TG sheep in lanes 2 and 4–8. c Monocytes were isolated from four WT and four TG sheep and stained for the expression of the monocyte markers, CD14 and CD11b. DAPI was used to stain the cell nuclei. Scale bar: 100 μm. d Similarly, TLR4 expression in the TG and WT monocytes was examined by immunofluorescence microscopy. Scale bar: 100 μm. e The protein expression of TLR4 in monocytes was examined by western blotting. f Quantification of the data in (e). g The mRNA expression of TLR4 in monocytes was evaluated by qRT-PCR. Both the mRNA and protein expression levels of TLR4 were significantly higher in the TG monocytes than in the WT monocytes. All data are presented as the mean ± SD, n ≥ 3; *P < 0.05, **P < 0.01. GAPDH was used for normalization