full length cellular form 96 prt1 nrp1 functional variant origen sequence  (OriGene)

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: AAV8-Tlcd3b transgene expression was verified in the rAAV8-Tlcd3b –treated retina of Tlcd3b − / − mice. ( A , B ) Immunofluorescence staining of representative contralateral untreated LE of Tlcd3b − / − mouse retina and treated RE of Tlcd3b − / − mouse retina at 2 months postinjection performed on postnatal day 21. TLCD3B and FLAG staining was undetectable in the PBS-injected LE of Tlcd3b − / − mice, whereas in the treated RE, robust staining was detected in the outer segment, the inner segment, and the outer nuclear layer of the retina. DAPI staining was performed to stain for cell nuclei. Scale bar : ( A ) 500 µm, ( B ) 20 µm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; OPL, outer plexiform layer; OS, outer segment.

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Expressing, Immunofluorescence, Staining, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: ERG analysis demonstrated functional improvement in 7-month-old rAAV8-Tlcd3b –treated Tlcd3b − / − mice injected at ( A ) P21 and ( B ) P120. Two-way ANOVA tests on electroretinography on ( A ) P21- and ( B ) P120-injected 7-month-old mice showed unaffected scotopic a-wave ( P = 0.67 for P21-injected mice; P = 0.42 for P120-injected mice) and significantly elevated scotopic ( P = 0.01 for P21-injected mice; P = 0.03 for P120-injected mice) and photopic b-waves ( P = 0.003 for P21-injected mice; P = 0.008 for P120-injected mice) in AAV-treated RE ( blue lines ) compared with untreated, PBS-injected LE ( red lines ). In addition, results of untreated wild-type (WT) age-matched mice ( black line ) are included for comparison, and there was no significant difference between the a-wave and b-wave amplitudes of WT and AAV-injected mice for both P21- and P120-treated groups. Significance at each luminescence was determined by Student's t -test. Error bars denote the SEM. NS, not significant; wild-type versus PBS-injected (* P < 0.05) and PBS-injected versus AAV-treated (# P < 0.05). AAV-treated RE ( blue line ), PBS-treated LE ( red line ), and WT control ( black line ).

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Functional Assay, Injection, Comparison, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: Preservation of photoreceptor morphology in treated Tlcd3b − / − mice at 7 months old. Hematoxylin and eosin staining of paraffin-embedded retinal sections was performed to assess morphologic changes in the untreated LE and treated RE of 7-month-old Tlcd3b − / − mice that were injected at ( A ) P21 and ( B ) P120. Scale bar : 50 µm. The butterfly plot includes ONL thickness measured from 18 equally spaced positions along the vertical median of the retina collected 6 months after P21 injection ( C ) and 3 months after P120 injection ( D ). Each dot represents an individual data point plotted over mean ± SEM. Position 0 corresponds to the optic nerve head. Error bars denote the SEM. Wild-type versus AAV-injected (* P < 0.05) and PBS-injected versus AAV-treated (# P < 0.05). For both P21- and P120-treated mice, no significant difference between the ONL thickness of AAV-treated RE and age-matched WT controls. AAV-treated RE ( blue line ), PBS-treated LE ( red line ), and WT control ( black line ).

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Preserving, Staining, Injection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: Assessment of cone morphology preservation in 7-month-old rAAV8-Tlcd3b –treated Tlcd3b − / − mice. Immunofluorescent staining of cone arrestin ( red ; A , B ) and PNA ( green ; C , D ) on cross sections of representative contralateral untreated LE and treated RE of Tlcd3b − / − mouse retina at 6 months postinjection performed on ( A , C ) P21 and ( B , D ) 3 months postinjection performed on P120. ( E ) In 7-month-old P21- and P120-injected Tlcd3b − / − mice, PNA staining ( green ) shows loss of cones compared with wild type in retinal whole mounts. Scale bar : 20 µm. ( F ) Cones were counted in the dorsonasal (DN), dorsotemporal (DT), ventronasal (VN), and ventrotemporal (VT) regions of ( E ) PNA-stained ( green ) retinal whole mounts of treated RE ( n = 4) and untreated LE ( n = 4) and then expressed as average number of cones per mm 2 (mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001. IS, inner segment; OS, outer segment.

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Preserving, Staining, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: Long-term rescue efficiency of rAAV8-Tlcd3b –mediated gene therapy in Tlcd3b − / − mice. Two-way ANOVA analysis on ERG data demonstrated functional improvement in RE ( blue line ) compared with the LE ( red line ) of both ( A ) P21-injected and ( B ) P120-injected 1-year-old Tlcd3b − / − mice, as indicated by the significantly elevated scotopic ( P = 0.004 for P21-injected mice; P = 0.01 for P120-injected mice) and photopic b-waves ( P = 0.0001 for P21-injected mice; P = 0.006 for P120-injected mice) in the treated eyes compared with the untreated eyes. Significance at each luminescence was determined by Student's t -test. Error bars denote the SEM. Wild-type versus PBS-injected (* P < 0.05) and PBS-injected versus AAV-treated (# P < 0.05). Hematoxylin and eosin staining of paraffin-embedded retinal sections from 1-year-old treated Tlcd3b − / − mice injected at ( C ) P21 and ( E ) P120 demonstrated photoreceptor preservation in the treated RE compared to the untreated LE. The butterfly plot includes ONL thickness measured from 18 equally spaced positions along the vertical median of the retina collected ( D ) 6 months after P21 injection and ( F ) 3 months after P120 injection. Each dot represents an individual data point plotted over mean ± SEM. Cone arrestin staining of paraffin-embedded retinal sections from 1-year-old treated Tlcd3b − / − mice injected at ( G ) P21 and ( H ) P120 demonstrated cone photoreceptor preservation in the treated RE compared to the untreated LE. Position 0 corresponds to the optic nerve head. Error bars denote the SEM. Scale bar : 20 µm.

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Functional Assay, Injection, Staining, Preserving

Journal: Investigative Ophthalmology & Visual Science

Article Title: AAV8 -Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model

doi: 10.1167/iovs.63.3.11

Figure Lengend Snippet: Mass spectrometric analyses of ceramide species and sphingosine in the retina of Tlcd3b − / − mice that were treated with rAAV8-Tlcd3b gene therapy. All retinas were collected when the P21-injected mice reached 3 months old. Noninjected wild-type mice were used as the external control. The level of sphingolipids was normalized to the total protein content in each sample. All values are mean ± SEM, and significance was determined by Student's t -test. * P < 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: Full-length Tlcd3b cDNA (Origene #MC210825; NM_029978.1) was sequence-verified and amplified by PCR.

Techniques: Injection, Control