Ts of the dermis were found to have increased by both 4 hours post-injection, and this effect lasted through 24 hours post-injection. Faster inflammatory reactions were observed in transgenic animals than in control animals.DiscussionInflammation is a protective reaction that allows organisms to remove injurious stimuli and to initiate the healing process. Pathogen invasion itself can trigger innate immunity [13]. TLR4 has been identified as a main receptor for LPS. It has been found to be expressed in many types of cells. The TLR4 signaling pathway is involved in cytokine release, monocyte chemotaxis and cell infiltration that produce disease-resistant effects [14,15]. Recently, studies have shown that overexpression of TLR4 can increase resistance to disease in mice [16]. TLR4 mutant mice appeared to have little or no response to LPS stimulation [17]. In this study, TLR4 cDNA was cloned, and a universal promoter was selected to produce transgenic sheep. Southern blot analysisconfirmed that 28.26 of the offspring were positive. Results showed TLR4 was highly expressed in these transgenic sheep. LPS is a natural immune activator. It can activate the mononuclear-phagocytic system, causing the release of a large number of cytokines and inflammatory mediators through TLR4 pathway [18]. It has been demonstrated that endotoxemia, colitis, and other inflammatory responses are associated with LPSinduced TLR4 expression [19?1]. Results indicated that TLR4 overexpression sheep quickly responded to LPS stimulation and increase the release of inflammatory cytokines, expanding the period of inflammatory response. TNF-a, a cytokine involved in TLR4 pathway, is one of the primary agents of the inflammatory response. It is important for pathogen resistance and in balancing the internal environment. In this study, TLR4 overexpression enhanced the level of expression of IL-8, IL-6, and TNF-a. IL-6 acts by promoting the differentiation and infiltration of activated macrophages and by up-regulating the expression cell-adhesion molecules, during immune response [22]. Under LPS stimulation, TNF-a and IL-6 increasing of expression levels were necessary for immune response [23]. As shown in one previous study, IL-6 was downregulated 
in TLR4 knock-out mice [24]. The primary function of IL-8 was induction of chemotaxis in AN 3199 certain cells. It also activates neutrophils. Fibroblasts overexpressing TLR4 were able to response to low doses of LPS (1ng/mL, 10ng/mL). Meanwhile,Overexpression of Toll-Like Receptor 4 in SheepFigure 3. Pro-inflammatory cytokines expression of overexpression TLR4 in fetal sheep fibroblasts under LPS stimulation. A), B), and C) show the expression of IL-6, IL-8, and TNF-a under LPS (100 ng/mL) stimulation. D), E), and F) show expression of IL-6, IL-8, and TNF-a under LPS (1000 ng/mL) stimulation. In graph: C = subjects transfected with p3S-LoxP transfected fetal fibroblasts, subjects transfected with TLR4 = pTLR4-3S fetal fibroblasts. Data are means 6 SE. * Values within the same concentration of LPS differ significantly across different groups (P,0.05). doi:10.1371/journal.pone.0047118.gFigure 4. Production of TLR4 overexpression transgenic sheep by KS-176 microinjection. A) Southern blot analysis. Samples from positive individuals (numbered 1, 2, 3, 4, 5, and 6) are shown with 7 as a negative control. p1, p2 and p3 are samples of transgenic vectors, here used as positive controls. 22948146 B) Transcription of TLR4 in transgenic sheep. C) immunohistochemi.Ts of the dermis were found to have increased by both 4 hours post-injection, and this effect lasted through 24 hours post-injection. Faster inflammatory reactions were observed in transgenic animals than in control animals.DiscussionInflammation is a protective reaction that allows organisms to remove injurious stimuli and to initiate the healing process. Pathogen invasion itself can trigger innate immunity [13]. TLR4 has been identified as a main receptor for LPS. It has been found to be expressed in many types of cells. The TLR4 signaling pathway is involved in cytokine release, monocyte chemotaxis and cell infiltration that produce disease-resistant effects [14,15]. Recently, studies have shown that overexpression of TLR4 can increase resistance to disease in mice [16]. TLR4 mutant mice appeared to have little or no response to LPS stimulation [17]. In this study, TLR4 cDNA was cloned, and a universal promoter was selected to produce transgenic sheep. Southern blot analysisconfirmed that 28.26 of the offspring were positive. Results showed TLR4 was highly expressed in these transgenic sheep. LPS is a natural immune activator. It can activate the mononuclear-phagocytic system, causing the release of a large number of cytokines and inflammatory mediators through TLR4 pathway [18]. It has been demonstrated that endotoxemia, colitis, and other inflammatory responses are associated with LPSinduced TLR4 expression [19?1]. Results indicated that TLR4 overexpression sheep quickly responded to LPS stimulation and increase the release of inflammatory cytokines, expanding the period of inflammatory response. TNF-a, a cytokine involved in TLR4 pathway, is one of the primary agents of the inflammatory response. It is important for pathogen resistance and in balancing the internal environment. In this study, TLR4 overexpression enhanced the level of expression of IL-8, IL-6, and TNF-a. IL-6 acts by promoting the differentiation 
and infiltration of activated macrophages and by up-regulating the expression cell-adhesion molecules, during immune response [22]. Under LPS stimulation, TNF-a and IL-6 increasing of expression levels were necessary for immune response [23]. As shown in one previous study, IL-6 was downregulated in TLR4 knock-out mice [24]. The primary function of IL-8 was induction of chemotaxis in certain cells. It also activates neutrophils. Fibroblasts overexpressing TLR4 were able to response to low doses of LPS (1ng/mL, 10ng/mL). Meanwhile,Overexpression of Toll-Like Receptor 4 in SheepFigure 3. Pro-inflammatory cytokines expression of overexpression TLR4 in fetal sheep fibroblasts under LPS stimulation. A), B), and C) show the expression of IL-6, IL-8, and TNF-a under LPS (100 ng/mL) stimulation. D), E), and F) show expression of IL-6, IL-8, and TNF-a under LPS (1000 ng/mL) stimulation. In graph: C = subjects transfected with p3S-LoxP transfected fetal fibroblasts, subjects transfected with TLR4 = pTLR4-3S fetal fibroblasts. Data are means 6 SE. * Values within the same concentration of LPS differ significantly across different groups (P,0.05). doi:10.1371/journal.pone.0047118.gFigure 4. Production of TLR4 overexpression transgenic sheep by microinjection. A) Southern blot analysis. Samples from positive individuals (numbered 1, 2, 3, 4, 5, and 6) are shown with 7 as a negative control. p1, p2 and p3 are samples of transgenic vectors, here used as positive controls. 22948146 B) Transcription of TLR4 in transgenic sheep. C) immunohistochemi.
