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Deng S.,China Agricultural University | Li G.,China Agricultural University | Zhang J.,Tianjin Institute of Animal science | Zhang X.,Tianjin Institute of Animal science | And 6 more authors.
Theriogenology | Year: 2013

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 in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, 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. © 2013 Elsevier Inc.


PubMed | CAS Institute of Zoology, Northeast Agricultural University, Tianjin Institute of Animal science and China Agricultural University
Type: | Journal: Oxidative medicine and cellular longevity | Year: 2015

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.


PubMed | State Oceanic Administration, CAS Institute of Zoology, Beihang University, Tianjin Institute of Animal science and China Agricultural University
Type: | Journal: Oxidative medicine and cellular longevity | Year: 2015

Toll-like receptor 4 (TLR4) is an important sensor of Gram-negative bacteria and can trigger activation of the innate immune system. Increased activation of TLR4 can lead to the induction of oxidative stress. Herein, the pathway whereby TLR4 affects antioxidant activity was studied. In TLR4-overexpressing sheep, TLR4 expression was found to be related to the integration copy number when monocytes were challenged with lipopolysaccharide (LPS). Consequently, production of malondialdehyde (MDA) was increased, which could increase the activation of prooxidative stress enzymes. Meanwhile, activation of an antioxidative enzyme, glutathione peroxidase (GSH-Px), was increased. Real-time PCR showed that expression of activating protein-1 (AP-1) and the antioxidative-related genes was increased. By contrast, the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) were reduced. In transgenic sheep, glutathione (GSH) levels were dramatically reduced. Furthermore, transgenic sheep were intradermally injected with LPS in each ear. The amounts of inflammatory infiltrates were correlated with the number of TLR4 copies that were integrated in the genome. Additionally, the translation of -glutamylcysteine synthetase (-GCS) was increased. Our findings indicated that overexpression of TLR4 in sheep could ameliorate oxidative injury through GSH secretion that was induced by LPS stimulation. Furthermore, TLR4 promoted -GCS translation through the AP-1 pathway, which was essential for GSH synthesis.


PubMed | Benedictine University, CAS Institute of Zoology, Tianjin Institute of Animal science and China Agricultural University
Type: | Journal: Journal of animal science and biotechnology | Year: 2016

Brucella 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.In 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 (P>0.05). 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 (P<0.05). 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.Here 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.


PubMed | Shanxi Datong University, Tianjin Institute of Animal science and China Agricultural University
Type: Journal Article | Journal: PloS one | Year: 2015

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.


Yu J.,Nankai University | Zhao Q.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Sun M.,Nankai University | Zhao X.,Nankai University
Proceedings of the World Congress on Intelligent Control and Automation (WCICA) | Year: 2015

Donor cell injection is a significant technique in nuclear transfer and many other biological operations. Recent achievements in biology and biomedicine demonstrate great potential of these micromanipulation technologies. Traditionally, these operations are done manually, which require high professional skills and are of low repeatability. To overcome these disadvantages, automatic operation of this procedure is required. This paper presents a robotic donor cell injection process in Somatic Cell Nuclear Transfer (SCNT). In this process, some techniques including target somatic cell selection, motion control of the injection pipette and the somatic cell, and oocyte penetration are discussed. Finally, experiments are conducted, and the results show that the proposed operating process is capable of performing donor cell injection with comparable speed of human operators. Over 98% detection success rate and 3 um positioning error verify practicability and applicability of the robotic donor cell injection procedure. © 2014 IEEE.


Liu Y.,Nankai University | Chen D.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Sun M.,Nankai University | And 2 more authors.
Chinese Control Conference, CCC | Year: 2016

The zona pellucida (ZP) is an important component of the oocyte. The stiffness of ZP plays crucial roles in many applications, such as nuclear transplantations (NT), embryo micro-injections, and clone. While conventional methods usually evaluate the deformability of the oocytes assuming the oocytes in static equilibrium when deforming, a novel evaluation obtained from the dynamic deformation is utilized in this work. In this paper, a method based on the subpixel cell contour detection algorithm for evaluating the zona pellucida of the porcine oocytes' deformability is proposed. A subpixel cell contour detection algorithm is used to detect the edge of the porcine oocytes to get the deformation dynamic curve; a self-developed pneumatic micro-injection system is used to apply pressure on the oocyte to deform the oocyte, and a transfer junction is got to evaluate the deformability of the porcine oocytes. The R2 of the fitted model is 0.9477. © 2016 TCCT.


Zhao Q.,Nankai University | Wu M.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Qin Y.,Nankai University | And 4 more authors.
Review of Scientific Instruments | Year: 2013

This paper presents a novel micropipette aspiration (MA) method based on a common pneumatic micro-injection system. This method is the first to quantify the influence of capillary effect on aspiration pressure using a balance pressure model, and in return, uses the capillary effect to quantify the aspiration pressure. Subsequently, the seal between the cell and the micropipette is detected to judge and exclude the ineffective MA attempts. The rationality of the balance pressure model is validated by the designed micropipette-filling experiments. Through applied to elasticity-determination of the cells with different sizes, the feasibility and versatility of this MA method are proved. With abilities to quantify aspiration pressures and detect the seam between the cell and the micropipette, our method is expected to advance the application of the commercial pneumatic injector in the MA of cells. Moreover, with the quantified volume of the liquid entering into the micropipette during MA process, our method also has a potential applicability to the study of the permeability of the cell membrane in the future. © 2013 AIP Publishing LLC.


Yu J.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Sun M.,Nankai University | Zhao X.,Nankai University
Chinese Control Conference, CCC | Year: 2015

As an effective approach to manipulate intracellular material exchange, such as injection of transgenic solutions or donor cells into receptor cells, microinjection possesses the incomparable advantage of introducing precisely defined amounts of substances into the cytoplasm without altering other components of the cell. Accurate quantification is generally obtained by calibrating the relationship between the injection volume and injection pressure in advance. Shift in the injection pressure and difference of the initial condition causes large deviation of the results. This paper presents a control method that can achieve the required injection volume with fast speed and high stability based on the flow characteristics of fluids inside a micropipette. First, a targeted control strategy has been brought up to resolve the problem of difficulty in microfluid dynamics modeling and instability of conventional control methods in settling this issue. Next, gray moment operators are employed to locate target edge to subpixel values in order to obtain higher precision of the injection volume. At last, the proposed method is applied to the automated transgenic injection tasks. Regulating time is approximately from 6 to 12 seconds, control accuracy of the injection volume is 0.0182 picoliter, and average operating speed is 1.5 cells/min. © 2015 Technical Committee on Control Theory, Chinese Association of Automation.


PubMed | Nankai University and Tianjin Institute of Animal Science
Type: Journal Article | Journal: The Review of scientific instruments | Year: 2014

This paper presents a novel micropipette aspiration (MA) method based on a common pneumatic micro-injection system. This method is the first to quantify the influence of capillary effect on aspiration pressure using a balance pressure model, and in return, uses the capillary effect to quantify the aspiration pressure. Subsequently, the seal between the cell and the micropipette is detected to judge and exclude the ineffective MA attempts. The rationality of the balance pressure model is validated by the designed micropipette-filling experiments. Through applied to elasticity-determination of the cells with different sizes, the feasibility and versatility of this MA method are proved. With abilities to quantify aspiration pressures and detect the seam between the cell and the micropipette, our method is expected to advance the application of the commercial pneumatic injector in the MA of cells. Moreover, with the quantified volume of the liquid entering into the micropipette during MA process, our method also has a potential applicability to the study of the permeability of the cell membrane in the future.

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