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DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.
Subject(s)
Abscisic Acid , Acetates , Cloning, Molecular , Cold Temperature , Cyclopentanes , Dendrobium , Genetics , Gene Expression Regulation, Plant , Oxylipins , Plant Proteins , Genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Response Elements , Stress, Physiological , NicotianaABSTRACT
Objective To investigate the effect of hepatitis B virus X (HBx)protein on the apoptosis of placental trophoblastic cells and its potential mechanism.Methods A pcDNA3.1 expression vector of HBx gene was constructed and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After transfection for 48 h,RT-PCR and immunofluorescence analyses were made to detect HBx mRNA and protein expressions.Flow cytometry was used to detect the early apoptosis status of JEG-3 and HTR-8 cells.The expressions of PI3K and p-Akt were detected by immunofluorescence and Western blotting.Results After transfection for 48 h,RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expressions were detected in JEG-3 and HTR-8 cells.Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection (P <0.05).Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells (P < 0.05 ).Conclusion HBx gene can be transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After the transfection,the early apoptosis of JEG-3 and HTR-8 cells is reduced.Its inhibition on apoptosis is related to the activation of the PI3K/Akt signaling path-way.
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Objective To investigate the effect of hepatitis B virus X (HBx)protein on the apoptosis of placental trophoblastic cells and its potential mechanism.Methods A pcDNA3.1 expression vector of HBx gene was constructed and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After transfection for 48 h,RT-PCR and immunofluorescence analyses were made to detect HBx mRNA and protein expressions.Flow cytometry was used to detect the early apoptosis status of JEG-3 and HTR-8 cells.The expressions of PI3K and p-Akt were detected by immunofluorescence and Western blotting.Results After transfection for 48 h,RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expressions were detected in JEG-3 and HTR-8 cells.Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection (P <0.05).Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells (P < 0.05 ).Conclusion HBx gene can be transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After the transfection,the early apoptosis of JEG-3 and HTR-8 cells is reduced.Its inhibition on apoptosis is related to the activation of the PI3K/Akt signaling path-way.
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Mitochondria play a central role in the regulation of energy metabolism and signal transduction in eukaryotic cells. Although many fluorescent labeling strategies have been developed for mitochondrial studies, the methods that enable labeling efficiency assessment at the single-mitochondrion level are still lacking. By employing the unique advantages of high sensitivity flow cytometry ( HSFCM ) in the sensitive, rapid, and quantitative multiparameter analysis of individual mitochondria, here we examined the performance of several different mitochondrial labeling strategies from the perspectives of brightness, labeling ratio, and stability. Mitochondria isolated from HeLa cells transfected with pAcGFP1-Mito plasmid upon transient or stable transfections, and mitochondria directly labeled with MitoTracker Green or SYTO 62 were analyzed by a laboratory-built three-channel HSFCM. Upon the quantitative measurement of fluorescence brightness, it was found that the fluorescence intensity of green fluorescent protein ( GFP ) in mitochondria isolated from cells with stable transfection was about 17. 7-fold higher than the transient transfection ones, and was approximately two orders of magnitude brighter than mitochondria labeled with MitoTracker Green. On the other hand, the fluorescence signal of SYTO 62 labeling decreased upon washing, indicating its rapid dissociation rate. The strong fluorescence intensity and good labeling stability make stable transfection an efficient method to label mitochondria. The experimental results demonstrates that HSFCM provides a powerful analytical tool to assess the performance of mitochondrial fluorescence labeling via high throughput single mitochondria analysis.
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Aim The TRPV1 plasmid was transiently transfected into human embryonic kidney HEK 293T cells to establish the heterologous expression system of TRPV1-channel. Methods The transfection efficiency was confirmed under fluorescence mi-croscope and the TRPV1 protein expression was identified by u-sing Western blot, and the functional characteristics of the chan-nel were studied by using the method of confocal microscopy. Results The transfection rate could reach 40% ~50%; the transfected cells were found to have a clear band at the corre-sponding position that TRPV1 should be, which indicated that TRPV1 channel protein was expressed in the transfected cells. The confocal microscopy imaging result showed that the trans-fected HEK 293T cells were activated by TRPV1 channel ago-nist. Conclusion Transient transfection of HEK 293T cells with TRPV1 channel is successfully constructed and the heterol-ogous TRPV1 channel is verified to have normal calcium-media-ting function.
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ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.
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<p><b>OBJECTIVE</b>To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus (HBV) replication and expression in HBV transient transfection model in vitro.</p><p><b>METHODS</b>The eukaryotic expression plasmid pHBV1.1, which contains 1.1-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells.</p><p><b>RESULTS</b>After the transfection of plasmid pHBV1.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells (P<0.05), and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group (P<0.05); Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group (P<0.01); Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group (P<0.01).</p><p><b>CONCLUSIONS</b>The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.</p>
Subject(s)
Humans , Hep G2 Cells , Hepatitis B , Drug Therapy , Hepatitis B virus , Physiology , Phyllanthus , Plant Extracts , Pharmacology , Transfection , Virus ReplicationABSTRACT
Objective: To design and prepare siRNAs targeting ski gene and to observe its influence on the biological functions of HepG2 cells, such as proliferation, cell cycle, apoptosis, etc.. Methods: Three specific siRNAs of ski were designed and synthesized, and were transiently transfected into HepG2 via cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry. Results: All the 3 specific ski-siRNA(A, B, C) effectively inhibited the expression of ski gene, with ski-siRNA-B having the highest inhibition rate(70%). Furthermore, the ski expression had a decreasing tendency with the transfection time. The proliferation of HepG2 cells was markedly inhibited by ski-siRNAs(P<0.05); the number of cells at S stage was obviously decreased, being 2 folds that of the negative control group. Conclusion: The siRNA of ski gene can effectively induce growth inhibition of HepG2 cells and reduce cells of S stage, which is possibly through down-regulation of ski gene.
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Objective:To study the expression of cbf?1 on mouse MDPC-23 cell line,and discuss the role of cbf?1 in the regulation of dental embryo development. Methods:By transient transfection,immunofluorescence,and western blot,the expression of cbf?1 on mouse MDPC-23 cells were studied. Results:The control MDPC-23 cells didn't express cbf?1.After transient transfected for 48 h,cbf?l expression was significantlly increased.BMP_(2)could induce low level expression of cbf?1. Conclusion:cbf?l expression was significantly increased 48h after transient transfection.