Sheep Kidney Cells Research Articles

Effects of GABA on Oxidative Stress and Metabolism in High-Glucose Cultured Mongolian Sheep Kidney Cells.

The Mongolian sheep, emblematic of the Inner Mongolian grasslands, is renowned for its exceptional stress resistance and adaptability to harsh environments, drawing considerable attention. Recent research has unveiled the novel role of γ-aminobutyric acid (GABA) in combating oxidative stress. This investigation examined how GABA impacts renal-cortex and medulla cells from Mongolian sheep exposed to high-glucose stress conditions, utilizing gene expression analysis and non-targeted metabolomics. Elevated glucose levels significantly reduced the viability of Mongolian sheep renal cells and increased reactive oxygen species (ROS) levels. Conversely, the introduction of GABA notably enhanced cell viability, reduced ROS production, and stimulated the expression of antioxidant genes (e.g., Gpx, SOD, CAT) in the renal cortex. In the renal medulla, CAT expression increased, while Gpx gene expression showed mixed responses. Metabolomics analysis indicated that high-glucose exposure altered various metabolites, whereas GABA alleviated the metabolic stress induced by high glucose through modulating glycolysis and the tricarboxylic acid cycle. In Mongolian sheep renal cells, GABA effectively mitigated oxidative damage triggered by high-glucose stress by upregulating antioxidant genes and regulating metabolic pathways, revealing insights into its potential mechanism for adapting to extreme environments. This finding offers a fresh perspective on understanding the stress resilience of Mongolian sheep and may provide valuable insights for research across diverse disciplines.

CHARACTERISTICS OF THE EQUINE RHINOPNEUMONIA VIRUS AK-2011

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks, and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0 lg TCID50/cm3. Sequencing and polymerase chain reaction (PCR) analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPE) induced by the AK-2011 virus stain (dilution 101) in calf trachea and horse kidney cell cultures were stable from the 1st to 10th passages, with biological activity of 5.75-6.00 lg TCID50/cm3. CPE caused by the virus were apparent on days 2-3, further developed intensively, and extended to 60-80% of the cell monolayer on days 5-7. In calf kidney, sheep kidney, green monkey kidney, and bovine kidney cell cultures, the same changes were observed 1-2 days later. The changes were slow, and by day 7-10 CPE extended to no more than 30-50% of ​​the cell monolayer. An attenuated strain AK-2011 of equine rhinopneumonitis virus was obtained. It was considered a candidate for the manufacture of a vaccine for equine rhinopneumonitis. Thus, the attenuated strain AK-2011 of the equine rhinopneumonitis virus was characterized.

AK-2011 strain for the development of a vaccine against equine rhinopneumonitis.

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0lg TCID50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 101 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months.

MiR-200c-141 Enhances Sheep Kidney Cell Reprogramming into Pluripotent Cells by Targeting ZEB1.

Background and ObjectivesSheep-induced pluripotent stem cells (siPSCs) have low reprogramming efficiency, thereby hampering their use in biotechnology and agriculture. Several studies have shown that some microRNAs play an important role in promoting somatic reprogramming in mouse and human. In this study, we investigated the effect of miR-200c-141 on somatic reprogramming in sheep and explored the mechanism of promoting the reprogramming.Methods and ResultsThe lentivirus system driven by tetracycline (TET)-on carrying Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, hTERT, and SV40LT (OSKMNLST) could reprogram sheep kidney cells into pluripotent cells. Overexpression of miR-200c-141 in combination with OSKMNLST could significantly improve the efficiency of sheep iPSC generation (p<0.01). Sheep iPSCs derived from miR-200c-141 showed embryonic stem cell (ESC)-like pluripotent properties, were positive for alkaline phosphatase and some pluripotent markers by quantitative real-time PCR (qRT-PCR) and immunofluorescence, and were able to differentiate into three germ layers in vitro. Oar-miR-200c was transfected into HEK293FT cells and was able to target the zinc finger E-box-binding homeobox 1 (ZEB1) 3’UTR using dual luciferase reporting analysis. Overexpression of oar-miR-200c in SKCs significantly reduced the expression of ZEB1, but increased the expression of E-cadherin by qRT-PCR and western blotting analysis.ConclusionsThese results suggest that miR-200c-141 can promote the reprogramming of sheep somatic cells to iPSCs, and oar-miR-200c targeted ZEB1 3’UTR, significantly decreased expression of ZEB1, and increased expression of E-cadherin. Oar-miR-200c may improve the MET process by affecting the TGF-β signaling pathway, thus improving the efficiency of somatic cell reprogramming in sheep.

Recombinant T7 Phage with FMDV AKT-III Strain VP1 Protein is a Potential FMDV Vaccine.

The capsid protein (VP1) of the foot-and-mouth (FMD) AKT-III strain was expressed on the surface of the T7 phage capsid (AKT-T7 strain) and the potential of AKT-T7 strain as an FMD vaccine was evaluated. The AKT-T7 strain was successfully constructed and was not cytotoxic to BHK-21, MDBK, or sheep kidney cells. The AKT-T7 strain was well phagocytosed by mouse macrophages. Immunization of BALB/c mice revealed that animals were quickly induced and produced high levels of FMDV antibodies. Monitoring data indicated that FMDV antibody levels could be maintained at higher levels for longer periods of time. The AKT-T7 strain induced high levels of IFN-γ levels in mice with little effect on IL-4. The AKT-T7 induced the mice to produce FMDV antibodies, which has the advantage of phage and FMDV, and is a potential candidate for an FMD vaccine.

Roles of p53 and ASF1A in the Reprogramming of Sheep Kidney Cells to Pluripotent Cells.

Since the first report of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka, numerous attempts have been made to derive iPSCs from other species via the ectopic expression of defined factors. Sheep iPSCs (siPSCs) have significant potential for biotechnology and agriculture. Although several groups have described siPSCs, the reprogramming efficiency was extremely low. The exogenous transgenes could be not silenced in the iPSCs, which hampered their development and application. Here, we report that p53 knockdown and antisilencing function 1A (ASF1A) overexpression promoted iPSC generation from sheep kidney cells (SKCs). Compared with transduction with eight human defined transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, hTERT, and SV40LT), the additional introduction of p53 RNA interference (RNAi) and/or ASF1A in the presence of small-molecule compounds [vitamin C (Vc) and valproic acid (VPA)] greatly improved the efficiency of sheep iPSC generation. The siPSCs exhibited morphological features similar to mouse embryonic stem cells (ESCs) and were positive for alkaline phosphatase and, pluripotent marker genes (Oct4, Nanog, Sox2, Rex1, TRA-1-60, TRA-1-81, and E-cadherin). Furthermore, these cells exhibited a normal karyotype of 54 chromosomes and were able to differentiate into all three germ layers both in vitro and in vivo. Moreover, the exogenous genes were silenced in siPSCs when p53 small hairpin RNA (shRNA) and ASF1A were added. Our results may help to reveal the role of p53 and ASF1A in sheep somatic cell reprogramming and provide an efficient approach to reprogramming sheep somatic cells.

Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference

Bovine viral diarrhea virus (BVDV) should be a ubiquitous viral pathogen to the cattle and sheep industry. This pathogen is responsible for severe economic losses. We previously showed that plasmid-mediated dual short hairpin RNA (shRNA) efficiently inhibit BVDV replication in bovine kidney epithelial (MDBK) cells. In this study, we delivered the dual shRNA system to sheep fibroblasts and generated transgenic cell colonies. These transgenic fibroblasts were further used for somatic cell nuclear transfer (SCNT). Three lambs were born at full term, but perished soon after birth. Integration of shRNA into the genome of cloned sheep was confirmed by PCR and expression of shRNA in transgenic sheep was confirmed by real-time PCR. Kidney epithelial cells were isolated from transgenic sheep and challenged with multiple BVDV subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). The dual shRNA expressed in transgenic kidney epithelial cells significantly inhibited BVDV replication in a cross-resistance manner. Our results showed that transgenic RNAi might be a useful tool for preparation of transgenic animals with increased resistance to BVDV.

Cytogenetic and immunological study of mammalian hybrid cell lines

Two hybrid cell lines, KS-RL-3 (hybrid between TK− sheep kidney cells and rabbit lymphocytes) and CR-KS TK− (hybrid between rabbit β-cells and TK− sheep kidney cells), were assayed cytogenetically. It was shown that these hybrid cell lines were characterized by the presence of both sheep and rabbit chromosomes, with a number and structure which varied depending on the cell type and the number of passages. In some cases aberrant chromosomes were identified. The modal chromosome number was 121–135 (40.7%) in the KS-RL-3 cell line and 106–120 (51.6%) in the CR-KS TK− cell line. CR-KS TK− and KS-RL-3 cells were identified in the testicles and ear lobes of experimental animals during periods of 7 to 28 days after cell inoculation. Partial immunological tolerance of the hybrid cell lines was suggested.

The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses.

Bovine viral diarrhoea virus (BVDV) isolates infect cultured Madin-Darby bovine kidney (MDBK) cells as efficiently as sheep kidney cells. In contrast, border disease virus (BDV) propagates poorly in MDBK cells but infects sheep cells very efficiently. The envelope glycoprotein E2 has been shown to be essential for virus infectivity. To explore the potential role of E2 in pestivirus host range in cell cultures, we engineered a chimeric BVDV with the E2 coding region from BDV. As expected, the BVDV-E2(bdv) chimera retained the ability of BDV to multiply in sheep cells but experienced a remarkable reduction in its ability to propagate and form plaques in MDBK, a phenotype that is characteristic of the E2 donor, BDV31 virus. Control chimeric BVDV bearing a type II E2 demonstrated that the heterologous E2 does not impair replication in MDBK or lamb cells. These results establish a role for E2 in determining the tropism of a pestivirus in cell culture.

Accidental persistent infection of cell lines by Newcastle disease virus, showing three unusual features--defective neuraminidase, temperature sensitivity and intranuclear inclusions.

A persistent, defective infection by an unknown strain of Newcastle disease virus (NDV) appeared accidentally in established lines of pig, ox and sheep kidney cells. Virus particles released from the persistently infected cells were not infectious and were deficient in neuraminidase activity. Synthesis of some of the virus-specified proteins in the persistently infected cells was temperature-sensitive. Co-cultivation of mixed populations of carrier cells and healthy chick embryo cells induced cell fusion with the formation of multinucleate heterokaryons and intra-nuclear inclusions. The development of inclusions in the chicken nuclei was not accompanied by 'rescue' of infectious NDV.

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