Jinlong Zhang

Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages.

Many groups of Gram-negative bacteria cause diseases that are harmful to sheep. Toll-like receptor 4 (TLR4), which is critical for detecting Gram-negative bacteria by the innate immune system, is activated by lipopolysaccharide (LPS) to initiate inflammatory responses and oxidative stress. Oxidation intermediates are essential activators of oxidative stress, as low levels of free radicals form a stressful oxidative environment that can clear invading pathogens. NO is an oxidation intermediate and its generation is regulated by nitric oxide synthase (iNOS). Guanosine triphosphate cyclohydrolase (GCHI) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for the production of inducible iNOS. Previously, we made vectors to overexpress the sheep TLR4 gene. Herein, first generation (G1) of transgenic sheep was stimulated with LPS in vivo and in vitro, and oxidative stress and GCHI expression were investigated. Oxidative injury caused by TLR4 overexpression was tightly regulated in tissues. However, the transgenic (Tg) group still secreted nitric oxide (NO) when an iNOS inhibitor was added. Furthermore, GCHI expression remained upregulated in both serum and monocytes/macrophages. Thus, overexpression of TLR4 in transgenic sheep might accelerate the clearance of invading microbes through NO generation following LPS stimulation. Additionally, TLR4 overexpression also enhances GCHI activation.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin‐enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin‐enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1‐AANAT and pBC1‐ASMT were co‐injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty‐four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT‐PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin‐enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild‐type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Transgenic cloned sheep overexpressing ovine toll-like receptor 4

An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured inxa0vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured inxa0vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.

A 90-Day Toxicology Study of Meat from Genetically Modified Sheep Overexpressing TLR4 in Sprague-Dawley Rats

Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

BackgroundBrucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth.ResultsIn total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (Pu2009>u20090.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (Pu2009<u20090.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.ConclusionsHere we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Boar seminal plasma inhibits cryo-capacitation of frozen-thawed ram sperm and improves fertility following intracervical insemination

Cryopreservation has numerous deleterious effects on sperm structure and function, which can be reduced by adding seminal plasma (SP), either autologous or heterologous. The objective was to determine effects of adding boar SP to the freezing extender on sperm quality, tyrosine phosphorylation and fertilizing ability of frozen-thawed ram sperm. Semen was collected from eight Small-tail Han rams and extended in a glucose-egg yolk buffer supplemented with 0, 20, 40, or 60% porcine SP (from Large white boars). Compared to all other groups, 40% boar SP increased sperm viability and motility (Pxa0<xa00.05), whereas 20% boar SP had no beneficial effect, and 60% SP reduced sperm quality and motility (Pxa0<xa00.05). Compared to control (0%), 40% boar SP inhibited cryo-capacitation and tyrosine phosphorylation of frozen-thawed ram sperm, and improved the proportion of capacitated sperm and tyrosine phosphorylation after inxa0vitro capacitation (Pxa0<xa00.05). Furthermore, based on viscous medium penetration tests, 40% boar SP increased sperm penetration (944.7xa0±xa0121.5 vs 555.3xa0±xa088.7; Pxa0<xa00.05). Finally, 40% boar SP improved pregnancy rate for intracervical AI (47.5 vs 33.3%; Pxa0<xa00.05), apparently due to inhibition of cryo-capacitation, although pregnancy rate for intravaginal AI was not affected (31.6 vs 30.0%).

Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels

ABSTRACT To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with AANAT transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of AANAT and melatonin synthetic function than those PBMCs from the wild-type (WT) animals. After challenge with lipopolysaccharide (LPS), the transgenic PBMCs had increased autophagosomes and LC3B expression while they exhibited suppressed production of the proinflammatory cytokines, IL1B and IL12 (IL12A-IL12B/p70), compared to their WT. The mechanistic analysis indicated that the anti-inflammatory activity of endogenous melatonin was mediated by MTNR1B (melatonin receptor 1B). MTNR1B stimulation activated the MAPK14 signaling pathway to promote cellular macroautophagy/autophagy, thus, suppressing the excessive inflammatory response of cellular. However, when the intact animals challenged with LPS, the serum proinflammatory cytokines were significantly higher in the transgenic goats than that in the WT. The results indicated that endogenous melatonin inhibited the MAPK1/3 signaling pathway and ROS production, subsequently downregulated gene expression of BECN1, ATG5 in PMBCs and then suppressed the autophagy activity of PBMCs and finally elevated levels of serum proinflammatory cytokines in transgenic animals, Herein we provided a novel melatonin-enriched animal model to study the potential effects of endogenously produced melatonin on inflammatory responses and autophagy activity.

RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, Pu2009<u20090.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.

Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation

Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected from eighteen lambs which were randomly divided into control group (C) and melatonin group given an 18 mg melatonin implant subcutaneous (M). Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05), greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05). Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05). Furthermore, melatonin increased FI of FITC (global methylation level) on cumulus cells (p < 0.05). To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05) and higher methylation at two CpG sites of DNMT3a (p < 0.05), there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05). In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs.

Overexpression of Toll-like Receptor 4-linked Mitogen-activated Protein Kinase Signaling Contributes to Internalization of Escherichia coli in Sheep

Escherichia coli is one of the most common causal pathogens of mastitis in milk-producing mammals. Toll-like receptor 4 (TLR4) is important for host recognition of this bacteria. Increased activation of TLR4 can markedly enhance the internalization of E. coli. In this study, the relationship between TLR4 and mitogen-activated protein kinase (MAPK) signaling pathways in mediating E. coli internalization was evaluated in sheep monocytes. Using a TLR4-overexpressing transgenic (Tg) sheep model, we explored the bacterial internalization mechanism in sheep. We found that monocytes of Tg sheep could phagocytize more bacteria and exhibited higher adhesive capacity. The specific inhibition of p38 MAPK or c-Jun N-terminal kinase (JNK) or extracellular signal-regulated kinases (ERKs) reduced TLR4-dependent internalization of bacteria into sheep monocytes. Furthermore, the inhibition of MAPK signaling down-regulated the adhesive capacity of monocytes and the expression of scavenger receptors and adhesion molecules. Taken together, the overexpression of TLR4 in transgenic sheep enhanced the internalization of E. coli via MAPK signaling.