Search results for: β Galactosidase (CMV)
#31880951 2020/01/24 To Up
Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle.The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of β-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.
Laura Muraine, Mona Bensalah, Jamila Dhiab, Gonzalo Cordova, Ludovic Arandel, Alix Marhic, Maud Chapart, Stéphane Vasseur, Sofia Benkhelifa-Ziyyat, Anne Bigot, Gillian Butler-Browne, Vincent Mouly, Elisa Negroni, Capucine Trollet
2264 related Products with: Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle.100.00 ug500 100ug Lyophilized1 mg100 μg200 100 μg50ul1mg1 mg100 μg100.00 ug
#28210900 2017/02/16 To Up
False responses of Renilla luciferase reporter control to nuclear receptor TR4.Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.
Dongyun Zhang, Sam S Atlasi, Krishna K Patel, Zihao Zhuang, Anthony P Heaney
2080 related Products with: False responses of Renilla luciferase reporter control to nuclear receptor TR4.100 plates10 plates1 plate100 plates50 96TOne Vial: 5 X 10^6 Cells100 2 Pieces/Box10 plates100 96T
#28153047 2017/02/02 To Up
Arginase-I enhances vascular endothelial inflammation and senescence through eNOS-uncoupling.Augmented arginase-II (Arg-II) is implicated in endothelial senescence and inflammation through a mutual positive regulatory circuit with S6K1. This study was conducted to investigate whether Arg-I, another isoform of arginase that has been also reported to play a role in vascular endothelial dysfunction, promotes endothelial senescence through similar mechanisms.
Cuicui Zhu, Yi Yu, Jean-Pierre Montani, Xiu-Fen Ming, Zhihong Yang
2615 related Products with: Arginase-I enhances vascular endothelial inflammation and senescence through eNOS-uncoupling.16 Arrays/Slide1.00 flask2ug100ug Lyophilized8 Sample KitUp to 200 ml cultures2ug4 Arrays/Slide4 Sample Kit16-22 Sample Kit
#26751216 2016/01/11 To Up
Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.
Yoomi Choi, Min-Young Kang, Joung-Ho Lee, Won-Hee Kang, JeeNa Hwang, Jin-Kyung Kwon, Byoung-Cheorl Kang
2293 related Products with: Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.100 samples100μg30 isolations50 IU25 mg100ug Lyophilized100ug Lyophilized100ug250ul50ul100μg
#26656605 2015/12/11 To Up
A Novel Interaction between TFII-I and Mdm2 with a Negative Effect on TFII-I Transcriptional Activity.Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification.
Kateřina Cetkovská, Hana Šustová, Pavlína Kosztyu, Stjepan Uldrijan
1120 related Products with: A Novel Interaction between TFII-I and Mdm2 with a Negative Effect on TFII-I Transcriptional Activity.1 Set1 Set1 Set1 Set1 Set1 Set1 Set1 Set1 Set1 Set1 Set100ug Lyophilized
#26586434 2015/11/12 To Up
Adenoviral Vector Vaccination Induces a Conserved Program of CD8(+) T Cell Memory Differentiation in Mouse and Man.Following exposure to vaccines, antigen-specific CD8(+) T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8(+) T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8(+) T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8(+) T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.
Beatrice Bolinger, Stuart Sims, Leo Swadling, Geraldine O'Hara, Catherine de Lara, Dilair Baban, Natasha Saghal, Lian Ni Lee, Emanuele Marchi, Mark Davis, Evan Newell, Stefania Capone, Antonella Folgori, Ellie Barnes, Paul Klenerman
1853 related Products with: Adenoviral Vector Vaccination Induces a Conserved Program of CD8(+) T Cell Memory Differentiation in Mouse and Man.100ug Lyophilized100.00 ug1 kit96 tests100 μg0.1ml100 μg100ug Lyophilized1mg
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#23509359 2013/03/18 To Up
A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector.CD8(+) T cell memory inflation, first described in murine CMV (MCMV) infection, is characterized by the accumulation of high-frequency, functional Ag-specific CD8(+) T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of Ag is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus's low-level persistence and stochastic reactivation. We developed a new model of memory inflation based on a β-galactosidase (βgal)-recombinant adenovirus vector. After i.v. administration in C57BL/6 mice, we observed marked memory inflation in the βgal96 epitope, whereas a second epitope, βgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype, and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC class II. As in MCMV, only the inflating epitope showed immunoproteasome independence. These data define a new model for memory inflation, which is fully replication independent, internally controlled, and reproduces the key immunologic features of the CD8(+) T cell response. This model provides insight into the mechanisms responsible for memory inflation and, because it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans.
Beatrice Bolinger, Stuart Sims, Geraldine O'Hara, Catherine de Lara, Elma Tchilian, Sonja Firner, Daniel Engeler, Burkhard Ludewig, Paul Klenerman
1044 related Products with: A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector.24 tests50 μg96 tests20 25 Tests
#23251598 2012/12/12 To Up
Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.
Catherine E Winbanks, Claudia Beyer, Hongwei Qian, Paul Gregorevic
1466 related Products with: Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.200ul5 mg200ul1 mg
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