Zhengxing Lian

One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system

11 Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement,College of Animal Science and Technology, China Agricultural University, Beijing 100193, China2 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China3 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

Stronger In Vitro Phagocytosis by Monocytes-Macrophages Is Indicative of Greater Pathogen Clearance and Antibody Levels In Vivo

Monocytes-macrophages are crucial players in specific and nonspecific immune responses to protect organisms from invasion of bacteria or viruses. In this study, monocytes in circulation from 2 lines of Silky and Starbro chickens with different disease resistance were separated and cultured in vitro. After identification with acridine orange (AO), Giemsa staining, and CD14 immunostaining, monocytes-macrophages were used for adherence and phagocytosis test. The overall percentages of adherence of Silky monocytes was 1.5 times greater than that of Starbro (P < 0.01), which were 26.85% +/- 8.24% and 18.34% +/- 8.15%, respectively (mean +/- SD). The monocytes-macrophages phagocytic index, phagocytic product, and percentage of phagocytosis in Silkies were greater than in Star-bros, respectively. The difference of phagocytic index was significant (P < 0.05), that is, 3.70 +/- 1.75 and 1.97 +/- 0.31, respectively (mean +/- SD). Then, 20 Silkies were divided into 2 groups according to phagocytic index: high phagocytic index (HPI) group and low phagocytic index (LPI) group, to study the relationship between phagocytic activity in vitro and pathogen clearance. After being challenged against Salmonella Pullorum C79-13, the Silky birds with HPI produced a 3-fold greater level of specific antibodies compared with those with LPI (P < 0.01), 50.21 +/- 6.67 and 16.85 +/- 4.52, respectively (mean +/- SD). In contrast to LPI birds, HPI birds shed less Salmonella Pullorum bacteria (P < 0.05), that is, 168.98 x 10(8) +/- 294.74 x 10(8) compared to 385.40 x 10(8) +/- 399.94 x 10(8) (mean +/- SD), and the shedding peak of Salmonella Pullorum in the test span appeared 4 d earlier. These results indicated that phagocytosis of monocytes-macrophages had strong effects on antibody titer and bacteria shedding postchallenge, which could be used to predict the disease resistance in animals.

Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors

(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus–oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10−3, 10−5, 10−7, 10−9, and 10−11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10−7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

Effects of over-expression of TLR2 in transgenic goats on pathogen clearance and role of up-regulation of lysozyme secretion and infiltration of inflammatory cells

BackgroundToll-like receptor 2 (TLR2) is important to host recognition of invading gram-positive microbes. In goats, these microbes can cause serious mastitis, anthrax, tetanus, and other problems. Transgenic goats constitutively over-expressing TLR2 in many tissues serve as a suitable model for the study of the role of TLR2 over-expression in bacterial clearance.ResultsCapra hircus TLR2 over-expression vector (p3S-LoxP-TLR2) was used to generate transgenic goats by egg microinjection. The integration efficiency was 8.57%. Real-time PCR and immunohistochemical results confirmed that the goats over-expressing the TLR2 gene (Tg) expressed more TLR2 than wild-type goats (WT). Monocyte-macrophages from the bloodstreams of transgenic goats were stimulated with synthetic bacterial lipoprotein (Pam3CSK4) and by the promotion of interleukin-6 (IL-6) and IL-10 expression in vitro. The oxidative damage was significantly reduced, and lysozyme (LZM) secretion was found to be up-regulated. Ear tissue samples from transgenic goats that had been stimulated with Pam3CSK4 via hypodermic injection showed that transgenic individuals can undergo the inflammation response very quickly.ConclusionsOver-expression of TLR2 was found to decrease radical damage to host cells through low-level production of NO and MDA and to promote the clearance of invasive bacteria by up-regulating lysozyme secretion and filtration of inflammatory cells to the infected site.

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.

Metabolic properties of chicken embryonic stem cells

Cellular energy metabolism correlates with cell fate, but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood. Using a previously established chES cell model and electron microscopy (EM), we found that undifferentiated chES cells stored glycogen. Additionally, undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells, suggesting that chES cells direct glucose flux towards the glycogenic pathway. Moreover, we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway, as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs. Additionally, cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining, but it could be rescued by exogenous G6P. However, we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining. Moreover, differentiated chES cells expressed higher levels of GLUT1, HK1 and PFK mRNAs, while the level of GYS mRNA remained similar in control CEF cells. These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P, while differentiated chES cells have a decreased glycogen reserve, which suggests that the amount of glycogen is indicative of the chES cell state.

The screening and identification of endogenous retrovirus free CEMPs

The provirus DNA sequence of porcine endogenous retrovirus (PERV) distributed in the pig genome is the major obstacle that restricts the swine as the organ donors in xenotransplantation, and the copy number of PERV varies greatly among different breeds and individuals. In the experiment, 67 healthy, female Chinese Experimental Mini-Pigs (CEMPs) aged at 3–6 months were selected from the Animal Husbandry Station of China Agricultural University, the copy number of PERV and types of envelope protein gene (env) were then investigated by means of PCR analysis and Southern blotting. It is showed that the distribution of types of envelope protein gene in Landrace and CEMPs makes little difference, but the proportion of individuals carrying two types of envelope protein gene (env-A and env-B, which is denoted as env-AB) is much larger than those which carry only one type of envelope protein gene (env-A or env-B). Meanwhile, two endogenous retrovirus free pigs were found for the first time during our research, and the copy number of others is relatively low, which is about 10 to 20. All the results illuminate the genetic diversity of indigenous pig breeds in China and the potential of CEMPs to serve as organ donors in xenotransplantation.

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.

Four recombinant pluripotency transcriptional factors containing a protein transduction domain maintained the in vitro pluripotency of chicken embryonic stem cells

Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.

Single nucleotide polymorphism analysis on chicken extracelluar fatty acid binding protein gene and its associations with fattiness trait

Fattiness is an important parameter to estimate meat quality, which has high heritability. In this experiment, F2 chickens derived from Broilers crossing to Silky were used to study the effect of extracellular fatty acid binding protein (EX-FABP) gene on abdominal fat accumulation. 1.6 kb of the 5′ region of the gene was amplified by six pairs of primers, and then single nucleotide polymorphisms (SNPs) were detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. There were four nucleotides variations found, A-G at-1807, C-A at -1805, T-C at -1011 and a C insertion at -1000 respectively. The result of least square analysis suggests that the birds with BB genotype defined by the second pair of primer have a higher abdominal fat weight and abdominal fat percentage than the birds with the other genotypes (AA and AB). It implied that EX-FABP gene could be a candidate locus or linked to a major gene to significantly affect abdominal fat traits in chicken.