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#29045937   2017/10/18 Save this To Up

Adenovirus-Mediated Expression of BMP-2 and BFGF in Bone Marrow Mesenchymal Stem Cells Combined with Demineralized Bone Matrix For Repair of Femoral Head Osteonecrosis in Beagle Dogs.

This study investigated the effect of using adenovirus-mediated expression of bone morphogenetic protein 2 (Ad-BMP-2) and basic fibroblast growth factor (bFGF) in bone marrow mesenchymal stem cells (BMSCs) in combination with a demineralized bone matrix (DBM) to repair osteonecrosis of the femoral head (ONFH) in Beagle dogs.

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Bone marrow tumor and adj Bone marrow tumor and nor Human normal bone and ost Bone and cartilage cancer Bone cancer test tissue a Bone and cartilage tumor Bone Morphogenetic Protei Human Bone Morphogenetic Macrophage Colony Stimula Macrophage Colony Stimula Alkaline Phospatase (ALP) (7’-Benzyloxy-indolymet

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#28869733   2017/09/04 Save this To Up

[Effects of recombinant adenovirus Ad-miR-29b2c on HGC-27 cell proliferation and migration].

We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.

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#28836860   2017/08/24 Save this To Up

Enhancement or inhibition of PLCγ2 expression in rat hepatocytes by recombinant adenoviral vectors that contain full-length gene or siRNA.

We investigated the effects of recombinant adenovirus vectors that overexpress or silence PLCγ2 on the expression of this gene during hepatocyte proliferation. Hepatocytes were isolated, identified by immunofluorescent cytochemical staining and infected by previously constructed Ad-PLCγ2 and Ad-PLCγ2 siRNA1, siRNA2 and siRNA3. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy. Infection percentage was calculated by flow cytometry. mRNA and protein levels of PLCγ2 were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blotting, respectively. The viability of the infected hepatocytes was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We found that nearly 97% of cells were positive for the hepatocyte marker, CK18. After infection of Ad-PLCγ2 and Ad-PLCγ2 siRNA, more than 99% of hepatocytes expressed GFP significantly, and mRNA and protein expression of PLCγ2 was up-regulated significantly in Ad-PLCγ2 infected hepatocytes, but down-regulated in Ad-PLCγ2 siRNA2 infected cells. The cell proliferation rate decreased in PLCγ2-overexpressing cells, while the rate increased in PLCγ2-silencing cells. We verified that recombinant Ad-PLCγ2 and Ad-PLCγ2 siRNA2 were constructed successfully. These two recombinant vectors promoted or decreased the expression of PLCγ2 in rat hepatocytes and affected the cell proliferation rate, which provides a useful tool for further investigation of the role of PLCγ2 in hepatocyte apoptosis.

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#28827116   2017/08/22 Save this To Up

Targeting expression of antimicrobial peptide CAMA-Syn by adenovirus vector in macrophages inhibits the growth of intracellular bacteria.

Although purified and synthesized Cecropin A-magainin 2 (CAMA-syn) shows potent antibacterial activity in vitro, its ability to inhibit bacteria within mammal cells mediated by virus vector has not yet been investigated. To enhance its antimicrobial potential and reduce systemic side effects, it would be desirable to deliver CAMA-syn in macrophages by adenovirus vector. In this study,recombinant adenovirus Ad-MSP-CAMA/GFP were used to infect macrophages RAW264.7 cells in vitro and macrophages cells of lungs in vivo and the expression of CAMA-syn was detected by RT-PCR and observation of co-expression of GFP. Antimicrobial activity in cells was evaluated by colony enumeration. The results showed that expression of CAMA-syn in macrophages conferred antimicrobial activity against a series of bacteria, including E. coli and BCG(Bacillus Calmette-Guérin). To establish BCG infection animal model, 40 Kunming mice were randomly divided into the following four groups: adenoviral delivery of Ad-MSP-CAMA/GFP, Ad-CMV-CAMA/GFP, empty-virus Ad-GFP, and control PBS, respectively. The expression of CAMA-syn in mouse was confirmed by real-time PCR and GFP co-expression. In brief, 3 days after injection of adenoviral vector, mice were scarified, different tissues were sectioned and homogenized and colony-forming efficiency by these treated tissues was determined. The colony-forming efficiency of Ad-MSP-CAMA/GFP (78.31%) and Ad-CMV-CAMA/GFP (61.68%) showed significant reduction compared to control groups. No inhibition of bacterial colony was observed from tissues treated by the PBS or empty-virus control. In conclusion, our results demonstrated that macrophages-specific expression of antimicrobial peptide CAMA-syn in macrophages inhibited the growth of intracellular bacteria, providing a promise approach for the control of refractory intracellular infection.

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#28452633   2017/04/28 Save this To Up

Post-spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG.

OBJECTIVE The most actively explored therapeutic strategy for overcoming spinal cord injury (SCI) is the delivery of genes encoding molecules that stimulate regeneration. In a mouse model of amyotrophic lateral sclerosis and in preliminary clinical trials in patients with amyotrophic lateral sclerosis, the combined administration of recombinant adenoviral vectors (Ad5-VEGF+Ad5-ANG) encoding the neurotrophic/angiogenic factors vascular endothelial growth factor ( VEGF) and angiogenin ( ANG) was found to slow the development of neurological deficits. These results suggest that there may be positive effects of this combination of genes in posttraumatic spinal cord regeneration. The objective of the present study was to determine the effects of Ad5-VEGF+Ad5-ANG combination therapy on motor function recovery and reactivity of astrocytes in a rat model of SCI. METHODS Spinal cord injury was induced in adult Wistar rats by the weight-drop method. Rats (n = 51) were divided into 2 groups: the experimental group (Ad5-VEGF+Ad5-ANG) and the control group (Ad5-GFP [green fluorescent protein]). Recovery of motor function was assessed using the Basso, Beattie, and Bresnahan scale. The duration and intensity of infectivity and gene expression from the injected vectors were assessed by immunofluorescent detection of GFP. Reactivity of glial cells was assessed by changes in the number of immunopositive cells expressing glial fibrillary acidic protein (GFAP), S100β, aquaporin 4 (AQP4), oligodendrocyte transcription factor 2, and chondroitin sulfate proteoglycan 4. The level of S100β mRNA expression in the spinal cord was estimated by real-time polymerase chain reaction. RESULTS Partial recovery of motor function was observed 30 days after surgery in both groups. However, Basso, Beattie, and Bresnahan scores were 35.9% higher in the Ad5-VEGF+Ad5-ANG group compared with the control group. Specific GFP signal was observed at distances of up to 5 mm in the rostral and caudal directions from the points of injection. A 1.5 to 2.0-fold increase in the number of GFAP(+), S100β(+), and AQP4(+) cells was observed in the white and gray matter at a distance of up to 5 mm from the center of the lesion site in the caudal and rostral directions. At 30 days after injury, a 2-fold increase in S100β transcripts was observed in the Ad5-VEGF+Ad5-ANG group compared with the control group. CONCLUSIONS Intraspinal injection of recombinant adenoviral vectors encoding VEGF and ANG stimulates functional recovery after traumatic SCI. The increased number of S100β(+) astrocytes induced by this approach may be a beneficial factor for maintaining the survival and function of neurons. Therefore, gene therapy with Ad5-VEGF+Ad5-ANG vectors is an effective therapeutic method for SCI treatment.

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#28429670   2017/04/21 Save this To Up

Expression of Dominant Negative K6W-Ubiquitin in the Lens Epithelium via an Adenoviral Vector Delays Posterior Capsule Opacification in a Rabbit Model.

Ubiquitin is involved in cell proliferation and differentiation, and the objective of this study is to investigate the potential of dominant negative Ubiquitin (Ub) with a lysine to tryptophan mutation at the 6 position (K6W) through an adenoviral expression vector in the prevention of posterior capsule opacification (PCO) in a rabbit PCO model.

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Interleukin-34 IL34 (N-t Interleukin-34 IL34 anti Integrin â3 (Phospho Tyr Integrin â3 (Phospho Tyr Interferon-a Receptor Typ Rabbit Anti-Inf A Neurami Rabbit Anti-Influenza A H Rabbit Anti-Influenza A N Rabbit Anti-Influenza-A H Rabbit Anti-Influenza-A H Rabbit Anti-Influenza-A H Rabbit Anti-Influenza-A H

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#28297774   2017/03/15 Save this To Up

[Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells].

Objective: To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro. Methods: The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca(2+) concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups. Results: Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca(2+) compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). Conclusion: The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.

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#27882354   2016/11/24 Save this To Up

Bcl-2-associated athanogene 3 protects the heart from ischemia/reperfusion injury.

Bcl-2-associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9-expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.

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#27826623   2016/11/09 Save this To Up

[Corrigendum] Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/AKT pathway.

Following the publication of this article, an interested reader drew to our attention an anomaly associated with the presentation of the western blots in Fig. 4B and C. These contained the same GAPDH control data, even though Fig. 4B and C were intended to show the inhibition of osteosarcoma cell migration by a recombinant adenovirus (ad) expressing bone morphogenetic protein 9 (adBMP-9) in two different cell lines, MG-63 and HOS, respectively. After having re-examined our original data, we realize that the same GAPDH control bands were inadvertently selected for Fig. 4B and C. A corrected version of Fig. 4, containing alternative data obtained from the experiments performed in duplicate, is presented below. Western blot assay was performed to examine the effect of adBMP-9 on metalloproteinase‑9 (MMP-9) expression, showing that the expression of MMP-9 protein was significantly inhibited in the adBMP-9 group compared with the ad-green fluorescent protein (GFP) and the control (CON) groups in the MG-63 and HOS cell lines. In addition, no marked differences were noted between the ad-GFP group and the CON group. The error made with the selection of the control data did not affect the results in this study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused. [the original article was published in the International Journal of Oncology 41: 1809-1819, 2012; DOI: 10.3892/ijo.2012.1617].

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#27755951   2016/10/18 Save this To Up

Construction of recombinant adenovirus Ad-rat PLCγ2 and its effects on apoptosis of rat liver cell BRL-3A in vitro.

Although the role of PLCγ2 in apoptotic response has been reported, too little is known about whether PLCγ2 induces liver cell apoptosis during liver regeneration. Therefore, this study firstly packaged Ad-PLCγ2 recombinant adenovirus and primarily evaluated its effect on apoptosis of rat liver cell BRL-3A in vitro. Following ten days of co-transfection of pHBAd-MCMV-GFP-PLCγ2 and pHBAd-BHG into HEK293 cells, viral cytopathic effect (CPE) was apparent. Following three rounds of amplification, tissue culture infectious dose 50 (TCID50) assay showed that the titer value reached 1×1010 PFU/mL. After 24 h of transfection of recombinant adenovirus into BRL-3A cells, transfection efficiency of adenovirus into BRL-3A cells was above 90% when obsereved under fluorescent microscopy. qRT-PCR and Western blot assays showed mRNA and protein levels of PLCγ2 were significantly elevated in the transfected BRL-3A cells. Flow cytometric analysis showed that, compared with the control and Ad-GFP groups, cell apoptosis rate of Ad-PLCγ2 group were significantly increased (P<0.01), and the cell cycle in Ad-PLCγ2 group was arrested at G1 phase which was manifested by a marked increase (P<0.01) in the percentage of G1 phase cells and a great decrease (P<0.01) in the percentage of S and G2/M phase cells. It was concluded from above results that recombinant adenovirus Ad-PLCγ2 was packaged successfully, and could promote cell apoptosis by arresting the transition from G1 to S phase of BRL-3A cells.

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