aav2/5-cmv-hi-cre-gfp  (Addgene inc)

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Whole mount retinal immunohistochemistry showing AAV transduction in RGCs. (A) Confocal image of whole mounted retina showing transduction of AAV2-CMV-GFP. (B) Confocal image of whole mounted retina showing transduction of AAV2-shRNA- Pten -GFP. (C) Confocal image of flat mounted retina showing GFP expressing in RGCs. (D) RBPMS label RGCs specifically as a marker. (E) More than half of RGCs are transduced by AAV and express GFP; merge image. Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Immunohistochemistry, Transduction, shRNA, Expressing, Marker

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Downregulation of Sox11 facilitates RGC survival following ONC. (A) Sox11 is partially knocked out by intravitreal injection of AAV2-CMV-Cre-GFP in 4 to 6-week-old Sox11 f/f mice. The control group receives AAV2-CMV-GFP. Optic nerve crush is performed 2 weeks after viral vector injection. Twelve days after ONC, Alexa Fluor 647-conjugated cholera toxin B (CTB) is used to label regenerating axons. Two days later mice are sacrificed. (B) For the retinal whole mounts, RGCs are labeled with the pan-RGC marker RBPMS. The number of AAV2-GFP vector transduced RGCs without crush injury is 1782 ± 117.5/mm 2 . (C) Downregulation of Sox11 in RGCs greatly increases the survival of AAV2 vector transduced RGCs (double labeled by GFP and RBPMS) after ONC as compared to AAV2-CMV-GFP injection with ONC group (357 ± 7.1/mm 2 vs. 158 ± 15.7/mm 2 , n = 4, p = 0.029, Mann–Whitney U -test). (D) The number of total RGCs labeled by RBPMS without crush injury is 3389 ± 92.1/mm 2 . When comparing the total RGCs labeled by RBPMS, it shows that Sox11 partial knockout in the Sox11 f/f mice significantly increases RPBMS-positive RGC survival after ONC as compared to AAV2-CMV-GFP ONC group (1398 ± 63/mm 2 vs. 828 ± 23/mm 2 , n = 4, p = 0.028, Mann–Whitney U -test). Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Injection, Plasmid Preparation, Labeling, Marker, MANN-WHITNEY, Knock-Out

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Representative images of optic nerves showing regenerating axons from different groups at 2 weeks after injury. (A) Pseudocolor green was used for the CTB-labeled axons in all the optic nerve images of this study for clear visual observation (red asterisks represent the crush site). Quantification axon number at 0.5 mm (B) and 1 mm (C) from crush site reveals that there is a statistically significant increase in the number of axons following an injection of either AAV2-GFP or AAV2-Cre-GFP in the Sox11 f/f mice relative to ether the C57BL/6J control of the Sox11 f/f control. This indicates that the increased axon regeneration is due to the use of AAV2-gene delivery systems in the retina and not the partial knockout of Sox11 . A similar result was observed for the distance of longest five regenerating axons traveled (D) and the distance of longest single regenerating axons (E) . Again, the most regeneration was observed in the animals that received gene delivery in the retina by AAV2 vectors. There was a significant difference ( p < 0.05, n = 4, Mann–Whitney U -test) between the ONC only mice and the Sox11 f/f mice that received intravitreal injections of either AAV2-GFP or AAV2-Cre-GFP. No statistic difference was found between the two ONC only groups. No statistic difference was found between the Sox11 pKO and the GFP control groups. N = 4 per group. Scale bar = 200 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Labeling, Injection, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Genetics

Article Title: Different Effect of Sox11 in Retinal Ganglion Cells Survival and Axon Regeneration

doi: 10.3389/fgene.2018.00633

Figure Lengend Snippet: Downregulation of Sox11 facilitates RGC survival following optic nerve crush and regeneration experiment. (A) Sox11 and Pten are knocked down by intravitreal injection of AAV2-CMV-Cre-GFP or AAV2-shRNA- Pten -GFP in 4 to 6 week-old Sox11 f/f mice. The control group receives AAV2-CMV-GFP. Optic nerve crush is performed 2 weeks after viral vector injection. Twelve days after ONC, Alexa Fluor ® 647-conjugated CTB is used to label regenerating axons. Two days after the CTB injections the animals are sacrificed, the retinas and full-length optic nerves are removed. (B) RGCs are labeled with pan-RGC marker RBPMS in retinal whole mounts. (C) Downregulation of both Sox11 and Pten resulted in a significant increase in RGC survival as compared to Pten knockdown only (1393 ± 94/mm 2 vs. 722 ± 77/mm 2 , n = 4, ∗ p < 0.05, Mann–Whitney U -test). Scale bar = 100 μm.

Article Snippet: To downregulate Sox11, Sox11 f/f mice received intravitreal injections of AAV2-CMV-GFP-Cre [2 μl of 10 12 vg/mL, expressing Cre recombinase with GFP from a bicistronic vector (Addgene plasmid #49056), produced by Fred Gage ( )].

Techniques: Injection, shRNA, Plasmid Preparation, Labeling, Marker, MANN-WHITNEY