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Screening and stability of Madin-Darby bovine kidney cell strainco-expressing the capsid precursor protein P1-2A gene and the protease 3C gene of Foot-and-mouth Disease Virus
共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

Jiong Li,Yanhong Liu,Xiangtao Liu,Youjun Shang,Junlin Liu,Fanglan An,
李炯
,刘艳红,刘湘涛,尚佑军,刘俊林,安芳兰

微生物学报 , 2008,
Abstract: Abstract: Objective] To screen the Madin-Darby bovine kidney cell strains for stable expression of capsid protein of foot-and-mouth disease virus (FMDV ). Methods] We obtained two genes coding for capsid precursor protein (P1-2A) and protease (3C) of FMDV by PCR from recombinant plasmids pMD-P1-2A and pMD-3C.The recombinant retroviral vector pBABE-puro/P1-2A-3C was constructed by inserting P1-2A gene and 3C gene into pBABE-puro. Both the recombinant plasmid pBABE-puro/P1-2A-3C and the plasmid pVSV-G were co-transfected into packaging cells GP2-293 by liposome-mediated transfection method. As a result, the recombinant pseudovirus was produced. The pseu-dovirus infected the interesting target cell MDBK. The infected MDBK cells were selected by puromycin(2.5 mg/mL) for 2 weeks. The monoclonal cells were selected using cloning rings. The expression of capsid protein was detected by indirect immunofluorescence and sandwich-ELISA. The empty capsids of FMDV were observed under electron microscope. Re-sults] The capsid protein of FMDV was expressed in MDBK cells. The expression levels in screened cell strains of various passages showed no significant difference. Conclusion] The MDBK cell strains for stable expression capsid protein of FMDV were successfully screened, which laid a foundation of development of FMDV subunit vaccine.
Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells
Campos, A.H.;Schor, N.;
Brazilian Journal of Medical and Biological Research , 2000, DOI: 10.1590/S0100-879X2000000100015
Abstract: calcium oxalate (caox) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. the present study analyzes a series of mechanisms possibly involved in caox uptake by madin-darby canine kidney (mdck) cells. caox crystals were added to mdck cell cultures and endocytosis was evaluated by polarized light microscopy. this process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nm; n = 6; 43.9% inhibition; p<0.001) or thapsigargin (1 μm; n = 6; 33.3% inhibition; p<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 μm; n = 8; 46.1% inhibition; p<0.001) or cytochalasin b (10 μm; n = 8; 34.2% inhibition; p<0.001). furthermore, caox uptake was reduced when the activity of protein kinase c was inhibited by staurosporine (10 nm; n = 6; 44% inhibition; p<0.01), or that of cyclo-oxygenase by indomethacin (3 μm; n = 12; 17.2% inhibition; p<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 μm; n = 6), tetraethylammonium (1 mm; n = 6) or cromakalim (1 μm; n = 6). taken together, these data indicate that the process of caox internalization by renal tubular cells is similar to the endocytosis reported for other systems. these findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.
Effects of different concentrations of artemisinin and artemisinin-iron combination treatment on Madin Darby Canine Kidney (MDCK) cells
Amir Ali Shahbazfar, Payman Zare, Hemn Mohammadpour, Hossein Tayefi-Nasrabadi
Interdisciplinary Toxicology , 2012, DOI: 10.2478/v10102-012-0006-5
Abstract: Artemisinin is a sesquitrepenelactone with an endoperoxide bridge. It is a naturally occurring substance from Artemisia species plants. Artemisia species have been used in oriental medicine for centuries to treat malaria, gastrointestinal helminthosia, diarrhea, and as an antipyretic and sedative agent. Antileishmanial activity of the plants has been announced a few years ago. Dogs are the most important reservoir of leishmaniasis in some parts of the world. To use it as an antileishmanial drug in dogs, its side effects on different organs, among them the kidney as the organ of elimination have to be elucidated. Artemisinin with different concentrations (0.15, 0.3, 0.6 and 1.2 μg/ml) was added to the culture of MDCK (Madin darby canine kidney) cells with and without iron (86 μg/dl). All the changes were controlled and photographed every 12 hours using an invert microscope. After 60 hours, supernatants and cell extracts were examined for LDH (lactate dehydrogenase) concentration and total protein. Also TBARS (thiobarbituric acid reactive substances) test was performed on cell extracts. Some microscopic slides were prepared from the cells and stained with hematoxylin-eosin for microscopic exams. Biochemical parameters showed cellular reaction and injury in a concentration dependent manner. Cell injury was more severe in the iron-added groups. Microscopic exams showed cell and nuclear swelling, granular degeneration, vacuole and vesicle formation, cellular detachment, piknosis, karyorrhexis, cellular necrosis and inhibition of new mitosis. On using the drug for leishmaniasis treatment in the dog, it should be done with caution and supervision.
Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells  [cached]
Campos A.H.,Schor N.
Brazilian Journal of Medical and Biological Research , 2000,
Abstract: Calcium oxalate (CaOx) crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively) to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK) cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9% inhibition; P<0.001) or thapsigargin (1 μM; N = 6; 33.3% inhibition; P<0.005) administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 μM; N = 8; 46.1% inhibition; P<0.001) or cytochalasin B (10 μM; N = 8; 34.2% inhibition; P<0.001). Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44% inhibition; P<0.01), or that of cyclo-oxygenase by indomethacin (3 μM; N = 12; 17.2% inhibition; P<0.05); however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 μM; N = 6), tetraethylammonium (1 mM; N = 6) or cromakalim (1 μM; N = 6). Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.
High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7  [PDF]
Itsuki Hamamoto, Hiroshi Takaku, Masato Tashiro, Norio Yamamoto
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059892
Abstract: Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.
N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells  [PDF]
Berta Puig, Hermann C. Altmeppen, Dana Thurm, Markus Geissen, Catharina Conrad, Thomas Braulke, Markus Glatzel
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024624
Abstract: The cellular prion protein (PrPC) plays a fundamental role in prion disease. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrPC is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrPC and also replaced the GPI-anchor of PrPC by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrPC in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrPC. Exchange of the PrPC GPI-anchor for the one of Thy-1 redirects PrPC to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrPC, with the GPI-anchor being dominant over N-glycans.
Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions  [PDF]
Yong Quan,Yisheng Jin,Teresa N. Faria,Charles A. Tilford,Aiqing He,Doris A. Wall,Ronald L. Smith,Balvinder S. Vig
Pharmaceutics , 2012, DOI: 10.3390/pharmaceutics4020314
Abstract: The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell ? membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix ? Canine GeneChip ?. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter ( MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell ? membranes. When cells were differentiated on Transwell ? membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5–7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.
Specific Cell Cycle Synchronization with Butyrate and Cell Cycle Analysis by Flow Cytometry for Madin Darby Bovine Kidney (MDBK) Cell Line
C.J. Li,T.H. Elsasser
Journal of Animal and Veterinary Advances , 2012,
Abstract: We investigated the property of the cell cycle arrest induced by butyrate and compared cell synchronization induced by butyrate, serum deprivation and the combination of serum deprivation and aphidicolin. The site of growth inhibition and cell cycle arrest was studied by BrdU incorporation and flow cytometry analyses. By combining BrdU incorporation and DNA content analysis, not only can the overlapping between different cell populations be eliminated, but also the frequency and nature of individual cells that have synthesized DNA can be determined. Exposure of MDBK cells to 10 mM butyrate caused inhibition of growth and cell cycle arrest in a reversible manner. Evidence is presented that butyrate affected the cell cycle at a specific point immediately after mitosis and at a very early stage of the G1 phase. After release from butyrate arrest, MDBK cells underwent synchronous cycles of DNA synthesis and transited through the S phase. The results showed that most serum deprivation treated cells were arrested at the G1 phase and aphidicolin treated cells were arrested at the early S phase. The effects of the three treatments were reversible. Using butyrate-synchronized cells, we determined that it takes about 8 h for G1 synchronized cells, induced by butyrate, to progress into the S phase and 8 h for the completion of the S phase. One cycle of cell division for MDBK cells is about 20 h.
The Correlation of IFN γ to the Preferential Isolation of Influenza Type B over Type A Viruses in Madin Darby Canine Kidney Cells  [PDF]
Timothy Byaruhanga, Bernard Bagaya, Joyce Namulondo, John Timothy Kayiwa, Barbara Namagambo, Nicholas Owor, Irene Nabukenya, Barnabas Bakamuntumaho, Julius Julian Lutwama
Open Journal of Medical Microbiology (OJMM) , 2017, DOI: 10.4236/ojmm.2017.71002
Abstract: The isolation of influenza viruses in Madin Darby Canine Kidney (MDCK) cells has shown preferential isolation of a great percentage of Influenza B strains at the first passage than Influenza A strains. During in vitro isolation of Influenza viruses, majority of type A viruses are not confirmed as positive isolates by Hemagglutination (HA) assay despite having higher virulence and pathogenicity versus influenza B viruses. This study investigated the differences in IFN-γ and IL-10 cytokines secreted by MDCK cells upon exposure to the viruses and thus provided possible answers as to why influenza type B can easily be isolated from MDCK cells compared to influenza A. Positive influenza viruses were inoculated onto MDCK cells. IFN-γ and IL-10 cytokines stimulated by the viruses in MDCK cells were measured by indirect ELISA at 1 hour, 12 hours, 48 hours and 72 hours post inoculation (pi). A total of 46 specimens, with 23 specimens from each virus type were analyzed. IFN-γ was significantly higher at 1 hour pi in MDCK cells for influenza type A at p value of 0.024 than type B. No statistical significance was observed in means of cytokine IL-10 between influenza type A and type B. The study may show that IFN-γ is correlated to the preferential isolation of influenza type B over type A viruses. Anti-inflammatory cytokines may not necessarily be playing a role in the preferential growth of influenza type B, a less virulent type over influenza type A in MDCK cells.
Mutant p53 Cooperates with Knockdown of Endogenous Wild-Type p53 to Disrupt Tubulogenesis in Madin-Darby Canine Kidney Cells  [PDF]
Yanhong Zhang, Wensheng Yan, Xinbin Chen
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0085624
Abstract: Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H) or R261H (human equivalent R273H) in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors (Snail-1, Slug and Twist) is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.
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