Effects of Leonurine on oocyte maturation and parthenogenetic embryo development in sheep.

Leonurine (LEO), an alkaloid isolated from Leonurus spp., has anti-oxidant, anti-inflammatory and anti-apoptotic effects and can prevent damage caused by reactive oxygen species (ROS). These properties suggest that it can improve the maturation rate of oocytes and developmental ability of embryos, which are key parameters in animal breeding. In this study, the effects of LEO on in vitro maturation and early embryonic development in sheep oocytes were evaluated. Among various doses examined (0, 10, 20 and 40 µM), a dose of 20 µM was optimal with respect to the oocyte maturation rate. Compared with estimates in the control group, GSH levels and mitochondrial membrane potential of sheep oocytes treated with 20 µM LEO were significantly higher, and 40 µM LEO would affect oocyte maturation. Additionally, ROS levels were significantly lower, expression levels of the antioxidant genes CAT and SOD1 were significantly higher, and there was no significant difference in GPX3 expression. The Bax/Bcl-2 ratio and Caspase-3 expression were significantly reduced in the 20 µM LEO group. During early embryonic development in vitro, the cleavage rate and blastocyst rate were significantly higher in the 20 µM LEO treatment group compared to other groups. GSH levels and mitochondrial membrane potential were significantly higher, while ROS levels were significantly lower, and expression levels of the antioxidant genes CAT, GPX3 and SOD1 were significantly higher in eight-cell embryos treated with 20 µM LEO than in the control group. The Bax/Bcl-2 ratio and Caspase-3 levels were significantly decreased. In summary, LEO can reduce the effect of oxidative stress, improve the oocyte maturation rate and enhance embryonic development.

Peroxisome Proliferator-Activated Receptor γ Regulates Lipid Metabolism in Sheep Trophoblast Cells through mTOR Pathway-Mediated Autophagy.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key nuclear receptor transcription factor that is highly expressed in trophoblastic cells during embryonic attachment and is accompanied by rapid cell proliferation and increased lipid accumulation. We previously showed that the autophagy pathway is activated in cells after activation of PPARγ, accompanied by increased lipid accumulation. In this study, we used PPARγ agonist rosiglitazone and inhibitor GW9662, as well as autophagy activator rapamycin and inhibitor 3-methyladenine, to unravel the probable mechanism of PPARγ engaged in lipid metabolism in sheep trophoblast cells (STCs). After 12 h, 24 h, and 48 h of drug treatment, the levels of autophagy-related proteins were detected by Western blot, the triglyceride content and MDA level of cells were detected by colorimetry, and the lipid droplets and lysosomes were localized by immunofluorescence. We found that PPARγ inhibited the activity of mammalian target of rapamycin (mTOR) pathway in STCs for a certain period of time, promoted the increase of autophagy and lysosome formation, and enhanced the accumulation of lipid droplets and triglycerides. Compared with cells whose PPARγ function is activated, blocking autophagy before activating PPARγ will hinder lipid accumulation in STCs. Pretreatment of cells with rapamycin promoted autophagy with results similar to rosiglitazone treatment, while inhibition of autophagy with 3-methyladenine reduced lysosome and lipid accumulation. Based on these observations, we conclude that PPARγ can induce autophagy by blocking the mTOR pathway, thereby promoting the accumulation of lipid droplets and lysosomal degradation, providing an energy basis for the rapid proliferation of trophoblast cells during embryo implantation. In brief, this study partially revealed the molecular regulatory mechanism of PPARγ, mTOR pathway, and autophagy on trophoblast cell lipid metabolism, which provides a theoretical basis for further exploring the functional regulatory network of trophoblast cells during the attachment of sheep embryos.

FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation.

Dynamic changes in the endometrium are crucial for establishing early pregnancy in ruminants. Blastocyst elongation and implantation require hormones and nutrients to be secreted from the maternal endometrium. The fatty acid-binding protein FABP4 is a widely expressed fatty acid transport protein that promotes cell proliferation, migration, and invasion and is involved in conceptus implantation. However, the mechanism underlying the functional regulation of endometrial epithelial cells (EECs) by FABP4 during ovine peri-implantation remains unclear. We simulated hormonal changes in vitro in sheep EECs (SEECs) during the peri-implantation period and found that it elevated FABP4 expression. FABP4 inhibition significantly reduced cell migration, endoplasmic reticulum stress, and autophagy, suggesting that FABP4 regulates endometrial function in sheep. Moreover, the FABP4 inhibitor BMS309403 counteracted hormone-mediated functional changes in SEECs, and an endoplasmic reticulum stress activator and autophagy inhibitor reversed the abnormal secretion of prostaglandins induced by FABP4 inhibition. These results suggest that FABP4 affects ovine endometrial function during early gestation by regulating endoplasmic reticulum stress and autophagy in SEECs.

PPARγ regulates lipid metabolism and viability of sheep trophoblast cells.

Peroxisome proliferator-activated receptor γ (PPARγ) is highly expressed in trophoblast tissues in pregnancy during which the protein participates in diverse events, including embryo implantation and placental formation. However, little is known about the role of PPARγ in embryonic development. This study investigated the function of PPARγ in sheep trophoblast cells. The coding sequence of sheep PPARγ encoded 475 amino acids and included one synonymou mutation compared with the sheep reference sequence for PPARγ. The PPARγ protein was localized in the nucleus and cytoplasm of sheep trophoblasts. The relative expression of PPARγ was elevated in cells treated with rosiglitazone and reduced following administration of GW9662. Activation of PPARγ promoted cell proliferation and mobility, but inhibited apoptosis. In addition, stimulation of PPARγ promoted the expression of lipid metabolism-related genes FABP4 and PLIN2. The expression of prostaglandin metabolism-related genes PLA2G4A, PTGS2 and PTGES also was upregulated significantly in trophoblast cells when PPARγ was activated. In contrast, activation of PPARγ did not impact expression of the prostaglandin-related genes PGFS and SLCO2A1. At the same time, activation of PPARγ activity increased the ratio of PGE2 to PGF2α. Furthermore, fluorescence labelling showed that the numbers of cell lipid droplets increased after stimulation of PPARγ activity, but decreased when PPARγ was inhibited. In conclusion, PPARγ is critical for the regulation of lipid metabolism and prostaglandin synthesis and secretion in sheep trophoblast cells and also has a potent effect on cell proliferation and viability.

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer.

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine ß-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.

Examining the function of macrophage oxidative stress response and immune system in glioblastoma multiforme through analysis of single-cell transcriptomics.

Background: Glioblastoma (GBM), a prevalent malignant neoplasm within the neuro-oncological domain, has been a subject of considerable scrutiny. Macrophages, serving as the principal immunological constituents, profoundly infiltrate the microenvironment of GBM. However, investigations elucidating the intricate immunological mechanisms governing macrophage involvement in GBM at the single-cell level remain notably limited. Methods: We conducted a comprehensive investigation employing single-cell analysis, aiming to redefine the intricate cellular landscape within both the core and peripheral regions of GBM tumors. Our analytical focus extended to the profound study of macrophages, elucidating their roles within the context of oxidative stress, intercellular information exchange, and cellular trajectories concerning GBM and its assorted subpopulations. We pursued the identification of GBM prognostic genes intricately associated with macrophages. Utilizing experimental research to investigate the relevance of MANBA in the context of GBM. Results: Our investigations have illuminated the central role of macrophages in the intricate interplay among various subpopulations within the GBM microenvironment. Notably, we observed a pronounced intensity of oxidative stress responses within macrophages when compared to their GBM counterparts in other subpopulations. Moreover, macrophages orchestrated intricate cellular communication networks, facilitated by the SPP1-CD44 axis, both internally and with neighboring subpopulations. These findings collectively suggest the potential for macrophage polarization from an M1 to an M2 phenotype, contributing to immune suppression within the tumor microenvironment. Furthermore, our exploration unearthed GBM prognostic genes closely associated with macrophages, most notably MANBA and TCF12. Remarkably, MANBA appears to participate in the modulation of neuroimmune functionality by exerting inhibitory effects on M1-polarized macrophages, thereby fostering tumor progression. To bolster these assertions, experimental validations unequivocally affirmed the promotional impact of MANBA on GBM, elucidated through its capacity to curb cell proliferation, invasiveness, and metastatic potential. Conclusion: These revelations represent a pivotal step towards unraveling the intricate immunological mechanisms governing the interactions between macrophages and diverse subpopulations within the GBM milieu. Furthermore, they lay the foundation for the development of an innovative GBM prognostic model, with MANBA at its epicenter, and underscore the potential for novel immunotherapeutic targets in the ongoing pursuit of enhanced treatment modalities for this formidable malignancy.

PPARγ/mTOR Regulates the Synthesis and Release of Prostaglandins in Ovine Trophoblast Cells in Early Pregnancy.

Trophoblast cells synthesize and secrete prostaglandins (PGs), which are essential for ruminants in early gestation to recognize pregnancy. Hormones in the intrauterine environment play an important role in regulating PGs synthesis during implantation, but the underlying mechanism remains unclear. In this study, co-treatment of sheep trophoblast cells (STCs) with progesterone (P4), estradiol (E2), and interferon-tau (IFN-τ) increased the ratio of prostaglandin E2 (PGE2) to prostaglandin F2α (PGF2α) and upregulated peroxisome proliferator-activated receptor γ (PPARγ) expression, while inhibiting the mechanistic target of rapamycin (mTOR) pathway and activating cellular autophagy. Under hormone treatment, inhibition of PPARγ activity decreased the ratio of PGE2/PGF2α and cellular activity, while activating expression of the mTOR downstream marker-the phosphorylation of p70S6K (p-p70S6K). We also found that the PPARγ/mTOR pathway played an important role in regulating trophoblast cell function. Inhibition of the mTOR pathway by rapamycin increased the ratio of PGE2/PGF2α and decreased the expression of apoptosis-related proteins after inhibiting PPARγ activity. In conclusion, our findings provide new insights into the molecular mechanism of prostaglandin regulation of trophoblast cells in sheep during early pregnancy, indicating that the PPARγ/mTOR pathway plays an important role in PGs secretion and cell viability.

Single-Cell RNA Sequencing Reveals Cellular and Transcriptional Changes Associated With Traumatic Brain Injury.

Traumatic brain injury (TBI) is currently a substantial public health problem and one of the leading causes of morbidity and mortality worldwide. However, the cellular and transcriptional changes in TBI at single-cell level have not been well characterized. In this study, we reanalyzed a single-cell RNA sequencing (scRNA-seq) dataset of mouse hippocampus to identify the key cellular and transcriptional changes associated with TBI. Specifically, we found that oligodendrocytes were the most abundant cell type in mouse hippocampus, and detected an expanded astrocyte population, which was significantly activated in TBI. The enhanced activity of inflammatory response-related pathways in the astrocytes of TBI samples suggested that the astrocytes, along with microglia, which were the major brain-resident immune cells, were responsible for inflammation in the acute phase of TBI. Hormone secretion, transport, and exocytosis were found upregulated in the excitatory neurons of TBI, which gave us a hint that excitatory neurons might excessively transport and excrete glutamate in response to TBI. Moreover, the ependymal subpopulation C0 was TBI-specific and characterized by downregulated cilium movement, indicating that the attenuated activity of cilium movement following TBI might decrease cerebrospinal fluid flow. Furthermore, we observed that downregulated genes in response to candesartan treatment were preferentially expressed in excitatory neurons and were related to pathways like neuronal systems and neuroactive ligand-receptor interaction, indicating that candesartan might promote recovery of neurons after traumatic brain injury via mediating neuroactive ligand-receptor interactions and reducing excitotoxicity. In conclusion, our study identified key cell types in TBI, which improved our understanding of the cellular and transcriptional changes after TBI and offered an insight into the molecular mechanisms that could serve as therapeutic targets.

Establishment and characterization of a sheep endometrial epithelial cell line.

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

Application of Deep Learning Technology in Glioma.

A common and most basic brain tumor is glioma that is exceptionally dangerous to health of various patients. A glioma segmentation, which is primarily magnetic resonance imaging (MRI) oriented, is considered as one of common tools developed for doctors. These doctors use this system to examine, analyse, and diagnose appearance of the glioma's outward for both patients, i.e., indoor and outdoor. In the literature, a widely utilized approach for the segmentation of glioma is the deep learning-oriented method. To cope with this issue, a segmentation of glioma approach, i.e., primarily on the convolution neural networks, is developed in this manuscript. A DM-DA-enabled cascading approach for the segmentation of glioma, which is 2DResUnet-enabled model, is reported to resolve the problem of spatial data acquisition of insufficient 3D specifically in the 2D full CNN along with the core issue of memory consumption of 3D full CNN. For gliomas segmentation at various stages, we have utilized multiscale fusion approach, attention, segmentation, and DenseBlock. Moreover, for reducing three dimensionalities of the Unet model, a sampling of fixed region is used along with multisequence data of the glioma image. Finally, the CNN model has the ability of producing a better segmentation of tumor preferably with minimum possible memory. The proposed model has used BraTS18 and BraTS17 benchmark data sets for fivefold cross-validation (local) and online evaluation preferably official, respectively. Evaluation results have verified that edema's Dice Score preferable average, enhancement, and core areas of the segmentation of the glioma with DM-DA-Unet perform exceptionally well on the validation set of BraTS17. Finally, average sensitivity was observed to be high as well, which is approximately closer to the best segmentation model and its effect on the validation set of BraTS1 and has segmented gliomas accurately.

Identification and analysis of microRNAs-mRNAs pairs associated with nutritional status in seasonal sheep.

Given the important role of nutritional status for reproductive performance, we aimed to explore the potential microRNA (miRNA)-mRNA pairs and their regulatory roles associated with nutritional status in seasonal reproducing sheep. Individual ewes were treated with and without 0.3 kg/day concentrates, and the body condition score, estrus rate, and related miRNAs and target genes were compared. A total of 261 differentially expressed miRNAs were identified, including 148 hypothalamus-expressed miRNAs and 113 ovary-expressed miRNAs, and 349 target genes were predicted to be associated with nutritional status and seasonal reproduction in sheep. Ultimately, the miR-200b-GNAQ pair was screened and validated as differentially expressed, and a dual luciferase reporter assay showed that miR-200b could bind to the 3'-untranslated region of GNAQ to mediate the hypothalamic-pituitary-ovarian axis. Thus, miR-200b and its target gene GNAQ likely represent an important negative feedback loop, providing a link between nutritional status and seasonal reproduction in sheep toward enhancing reproductive performance and productivity.

[Construction of a general piggyBac transposon inducible cell immortalization vector and verification of its basic properties].

In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids.

Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line.

The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.

Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

Immortalized porcine intestinal epithelial cell cultures susceptible to porcine rotavirus infection.

In vitro studies related to various viral pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. The susceptibility to porcine rotavirus infection was evaluated by using a newly established porcine intestinal epithelial cell line. Immunohistochemical staining for cytokeratin confirmed that the cultured cells were epithelial cells. Measurement of cell viability and detection of infected cells confirmed that these epithelial cells were susceptible to porcine rotavirus infection. This study describes the cytopathic changes in cultured porcine intestinal epithelial cells during virus invasion. Following infection with porcine rotavirus, the cell cultures contained viral protein at 16 h post-infection as detected by direct immunofluorescence. The epithelial cell cultures provided competent target cells for studying host cell responses to porcine rotavirus and a homologous system for investigating the response of intestinal epithelial cells during viral infection.

Generation of mastitis resistance in cows by targeting human lysozyme gene to ß-casein locus using zinc-finger nucleases.

Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine ß-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in ß-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

PhiC31 integrase-mediated genomic integration and stable gene expression in the mouse mammary gland after gene electrotransfer.

BACKGROUND: PhiC31 integrase is capable of conferring long-term transgene expression in various transfected tissues in vivo. In the present study, we investigated the activity of phiC31 integrase in mouse mammary glands. METHODS: The normal mouse mammary epithelial cell line HC11 was transfected with FuGENE® HD Transfection Reagent (Roche Diagnostics, Shanghai, China). Transfection of the mouse mammary gland in vivo was performed by electrotransfer. Transgene expression was detected by western blotting and an enzyme-linked immunosorbent assay. Genomic integration and integration at mpsL1 was confirmed by a nested polymerase chain reaction. RESULTS: An optimal electrotransfer protocol for the lactating mouse mammary gland was attained through investigation of different voltages and pulse durations. PhiC31 integrase mediated site-specific transgene integration in HC11 cells and the mouse mammary gland. In addition, the site-specific integration occurred efficiently at the 'hot spot' mpsL1. Co-delivery of PhiC31 integrase enhanced and prolonged transgene expression in the mouse mammary gland. CONCLUSIONS: The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.

Establishment and evaluation of a stable cattle type II alveolar epithelial cell line.

Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs) also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells) by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II) that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.

Chicken hypersensitive site-4 insulator increases human serum albumin expression in bovine mammary epithelial cells modified with phiC31 integrase.

Achieving high expression levels of recombinant human serum albumin (HSA) for purification is a solution for the large amount of plasma-derived HSA needed in therapeutic applications. Here, we employed phiC31 integrase system and chicken hypersensitive site-4 (cHS4) insulators to construct a HSA expression vector for high-level HSA expression. The phiC31 integrase system mediated efficient transgene integration in bovine mammary epithelial cells (bMECs). A preferred pseudo attP site, which had 38 % identity with the 39 bp wild-type attP sequence, was detected in six out of 55 bMEC colonies. Addition of the cHS4 insulator to the phiC31 integrase system resulted in 8-20-fold increases of HSA expression compared with that of using integrase alone. Moreover, the reverse-oriented cHS4 insulator in the phiC31 integrase system provided the optimal level of HSA expression in bMECs.

[Detection and sequences analysis of bovine hepatitis E virus RNA in Xinjiang Autonomous Region].

Reverse transcription-nested polymerase chain reaction (RT-nPCR) was used to detect HEV (Hepatitis E virus, HEV) RNA in dung or anus-swab samples collected from the cows with the positive anti-HEV antibodies in dairy farms in Xinjiang Autonomous Region. 7 of 60 (11.67%) cows were positive for HEV RNA in one farm. 1 of 31 (3.23%) cows was positive in the other farm. PCR amplification products were cloned, sequenced and analyzed. The result showed that the homology among the 8 bovine HEV ORF2 189bp nucleotide amplification sequences was 96.3%-100.0%, suggesting the same genotype. Compared with HEV genotype 1, 2, 3 and 4 corresponding 189 bp nucleotide sequences, the average homology was 78.5%-86.4%, 81.7%-83.8%, 79.1%-85.3% and 84.3%-95.8% respectively. The maximum homology between 8 nucleotide amplification sequences and one sequence of genotype 4 was 93.2%-95.8%. Based on the sequence of the nucleic acid fragments, a phylogenetic tree was constructed. It was illustrated that 8 bovine HEV ORF2 189bp nucleotide amplification sequences in this study and human C5 strain, swine swC3, swXJ strain belonged to genotype 4. The finding suggested that infection of HEV probably existed in the cow group in Xinjiang Autonomous Region and the cow might be a new animal host except swine in origin of HEV infection.