human sirt1 standard  (R&D Systems)

Journal: Biomolecules

Article Title: Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

doi: 10.3390/biom12060830

Figure Lengend Snippet: AAV7m8 transduced RGCs with high efficiency after single intravitreal injection. ( A ) Schematic representation of the AAV7m8 capsid and genome structure for enhanced transduction and RGC selective targeting. AAV7m8 was modified to drive the expression of the transgene (e.g., eGFP, SIRT1) under the control of the SNCG promoter. Transgene is placed between 2 internal terminal repeats (ITRs). ( B ) AAV7m8 administration is achieved through single injection of the AAV vector into the vitreous humor of the mouse eye. ( C ) Fluorescence micrographs of flat mounted preparation of mouse retina after intravitreal injection of the AAV7m8.SNCG.eGFP vector. RGCs were labeled with a monoclonal antibody against Brn3a. Merged eGFP (green) and Brn3a (red) signal is shown. ( D ) Representative fluorescence micrographs of retinal cross-section after intravitreal administration of AAV7m8.SNCG.eGFP and immunostaining with Brn3a antibody. Note that cells expressing SNCG promoter-driven eGFP are localized mainly in the ganglion cell layer (GCL). Cells within the inner nuclear layer (INL) and outer nuclear layer (ONL) were not transduced with the AAV vector. ( E ) Quantification of the population of RGCs transduced with the AAV7m8.SNCG.eGFP vector. Data shown are means ± SEM ( n = 4). ( F ) Representative fluorescence micrographs of retinal flat mounts after intravitreal administration of AAV7m8.SNCG.SIRT1 and dual immunostaining with antibodies against Brn3a and human SIRT1. ( G ) Quantification of the population of SIRT1 + RGCs labeled with the Brn3a antibody in the retina of mice injected with the AAV7m8.SNCG.SIRT1 vector. Data shown are means ± SEM ( n = 6).

Article Snippet: The AAV vectors AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1 used in this study consist of the AAV7m8 capsid (a gift from John Flannery, UC-Berkeley, CA, USA), the human SNCG promoter [ ], the cDNA encoding enhanced GFP (eGFP) protein or codon-optimized Human SIRT1 (transcript variant 1) cDNA (Origene, Rockville, MD, USA), and the bovine growth hormone (bGH) polyadenylation signal.

Techniques: Injection, Transduction, Modification, Expressing, Plasmid Preparation, Fluorescence, Labeling, Immunostaining

Journal: Biomolecules

Article Title: Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

doi: 10.3390/biom12060830

Figure Lengend Snippet: Effects of AAV7m8-mediated selective expression of SIRT1 in RGCs on visual function. Visual function was determined by OKR tracking using the Optometry apparatus and software. Measurements were performed prior to MOG immunization and once every 7 days for up to 42 days postimmunization in AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1-treated and non-treated control and EAE mice. Data are presented as means ± SEM. * p < 0.05, AAV7m8. SNCG.SIRT1 (control) versus either vehicle (EAE) or AAV7m8.SNCG.eGFP (EAE), and # p < 0.05, AAV7m8. SNCG.SIRT1 (EAE) versus AAV7m8. SNCG.eGFP. Statistical significance was determined at each time point by one-way ANOVA with Tukey’s multiple comparisons test (EAE) ( n = 13 for AAV7m8.SNCG.SIRT1 (control); n = 7 for vehicle (EAE); n = 8 for AAV7m8.SNCG.eGFP (EAE); n = 17 for AAV7m8.SNCG.SIRT1).

Article Snippet: The AAV vectors AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1 used in this study consist of the AAV7m8 capsid (a gift from John Flannery, UC-Berkeley, CA, USA), the human SNCG promoter [ ], the cDNA encoding enhanced GFP (eGFP) protein or codon-optimized Human SIRT1 (transcript variant 1) cDNA (Origene, Rockville, MD, USA), and the bovine growth hormone (bGH) polyadenylation signal.

Techniques: Expressing, Software

Journal: Biomolecules

Article Title: Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

doi: 10.3390/biom12060830

Figure Lengend Snippet: Effects of AAV7m8-mediated selective expression of SIRT1 in RGCs on RGC survival. ( A ) Representative immunofluorescence micrographs of RGC staining in the central retina of vehicle-, AAV7m8.SNCG.eGFP- and AAV7m8.SNCG.SIRT1-treated control and EAE mice. Scale bar, 50 μm. ( B ) The total number of labeled RGCs present in 12 standardized retinal fields was determined. The average number of surviving RGCs per total sampled area of retina of control and EAE mice is shown in the graph. Values are means ± SEM. * p < 0.05 by one-way ANOVA and Tukey’s multiple comparisons test ( n = 13 for AAV7m8.SNCG.SIRT1 (control); n = 7 for vehicle (EAE); n = 8 for AAV7m8.SNCG.eGFP (EAE); n = 17 for AAV7m8.SNCG.SIRT1).

Article Snippet: The AAV vectors AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1 used in this study consist of the AAV7m8 capsid (a gift from John Flannery, UC-Berkeley, CA, USA), the human SNCG promoter [ ], the cDNA encoding enhanced GFP (eGFP) protein or codon-optimized Human SIRT1 (transcript variant 1) cDNA (Origene, Rockville, MD, USA), and the bovine growth hormone (bGH) polyadenylation signal.

Techniques: Expressing, Immunofluorescence, Staining, Labeling

Journal: Biomolecules

Article Title: Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

doi: 10.3390/biom12060830

Figure Lengend Snippet: Effects of AAV7m8-mediated SIRT1 gene transfer in RGCs on optic nerve inflammation. ( A ) H&E staining of optic nerve longitudinal sections from vehicle-, AAV7m8.SNCG.eGFP- or AAV7m8.SNCG.SIRT1-treated mice are shown. Yellow arrows indicate small foci of inflammatory cell infiltrates in representative optic nerve photos. Optic nerve sections were imaged with 20× objective lens. Scale bar, 50 μm. ( B ) Inflammation scores determined by ratings of the number of infiltrating cell clusters. Data represented as mean ± SEM. * p < 0.05 by one-way ANOVA and Tukey’s multiple comparisons test ( n = 13 for AAV7m8.SNCG.SIRT1 (control); n = 7 for vehicle (EAE); n = 8 for AAV7m8.SNCG.eGFP (EAE); n = 17 for AAV7m8.SNCG.SIRT1). NS = not significant.

Article Snippet: The AAV vectors AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1 used in this study consist of the AAV7m8 capsid (a gift from John Flannery, UC-Berkeley, CA, USA), the human SNCG promoter [ ], the cDNA encoding enhanced GFP (eGFP) protein or codon-optimized Human SIRT1 (transcript variant 1) cDNA (Origene, Rockville, MD, USA), and the bovine growth hormone (bGH) polyadenylation signal.

Techniques: Staining

Journal: Biomolecules

Article Title: Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis

doi: 10.3390/biom12060830

Figure Lengend Snippet: Effects of AAV7m8-mediated SIRT1 gene transfer in RGCs on optic nerve demyelination. ( A ) Representative photos of optic nerve longitudinal sections showing LFB staining. Optic nerve sections were visualized with light microscopy with 20× objective lens. Scale bar, 50 μm. ( B ) LFB stained areas of optic nerve were quantified. Data represented as mean ± SEM. * p < 0.05 versus AAV7m8.SNCG.SIRT1-treated non-EAE control mice. # p < 0.05 versus AAV7m8.SNCG.eGFP sham-treated EAE mice by one-way ANOVA and Tukey’s HSD post-test ( n = 13 for AAV7m8.SNCG.SIRT1 (control); n = 7 for vehicle (EAE); n = 8 for AAV7m8.SNCG.eGFP (EAE); n = 17 for AAV7m8.SNCG.SIRT1).

Article Snippet: The AAV vectors AAV7m8.SNCG.eGFP and AAV7m8.SNCG.SIRT1 used in this study consist of the AAV7m8 capsid (a gift from John Flannery, UC-Berkeley, CA, USA), the human SNCG promoter [ ], the cDNA encoding enhanced GFP (eGFP) protein or codon-optimized Human SIRT1 (transcript variant 1) cDNA (Origene, Rockville, MD, USA), and the bovine growth hormone (bGH) polyadenylation signal.

Techniques: Staining, Light Microscopy