Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 2. Optic nerve crush strongly induced Ecel1 expression in the retinas of the mice. (A) qRT-PCR was performed to analyze the Ecel1 mRNA expression level, normalized to Gapdh mRNA (n ¼ 7–11 each group). (B) The protein level of Ecel1 in the retinas was examined with an immunoblot analysis. Beta actin was used as an internal control. (C) Retinal protein was immunolabeled with anti-Ecel1 with or without a blocking peptide. (D) Immunohistochemistry showed that Ecel1 was absent from the retinas of mice that underwent a sham operation, while Ecel1 protein was abundantly expressed in the GCL on day 4 after optic nerve crush. (E) Ecel1 protein was strikingly localized in the mouse GCL on day 4 after optic nerve crush and not in the other cell layers. (F) Histogram showing the number of Ecel1-positive cells in the GCL 4 days after optic nerve crush (n ¼ 4 each group). (G) Immunoreaction for Ecel1 was colocalized with RBPMS, a marker of the RGCs. Error bars denote standard deviation. NC, nerve crush. **P < 0.01, ***P < 0.001. Scale bar: 20 lm.

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunolabeling, Blocking Assay, Immunohistochemistry, Marker, Standard Deviation

Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 3. Ecel1 expression was strongly induced in damaged RGCs treated with vinblastine but not in those treated with NMDA. Relative expression levels of Thy-1.2 (A), Rbpms (B), and Ecel1 (C) in vinblastine-treated retinas, and Thy1 (D), Rbpms (E), and Ecel1 (F) in NMDA-treated retinas, were determined with quantitative RT-PCR and normalized to the Gapdh level. Error bars denote SD (vinblastine; n¼7–8, NMDA; n¼6–10 for each group at each time point). Vin, vinblastine. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Expressing, Quantitative RT-PCR

Journal: Investigative ophthalmology & visual science

Article Title: Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

doi: 10.1167/iovs.18-23784

Figure Lengend Snippet: FIGURE 4. Ecel1 knockdown with the CRISPR/Cas9 system promoted RGC loss after optic nerve crush. (A) Structure of the mouse Ecel1 gene; the 2nd exon is magnified. The mouse Ecel1 gene has 18 exons and 17 introns. The translation start codon (ATG) is located on the second exon. The guide RNA sequences for Ecel1 and for CFP as a control are shown in the box. (B) T7 Endonuclease 1 (T7E1) assay revealing genome editing at the Ecel1 locus in sorted mCherry-expressing cells from AAV2-mCherry and AAV2-CRISPR/Cas9-injected retinas. Asterisks indicated indel formation. (C) Mutation pattern sequencing of Ecel1 locus. (D) Immunoblotting with an anti-Ecel1 antibody in retinas injected with AAV2-CRISPR/Cas9 4 days after optic nerve crush. (E) Quantification of Ecel1 protein levels normalized with b-actin in (D) (n ¼ 3 each). (F) Immunostaining images for Ecel1 with AAV2-CRISPR/Cas9-Ecel1 treatment 4 days after optic nerve crush. (G) Histogram showing the number of Ecel1-positive cells in the GCL 4 days after optic nerve crush and treatment with AAV2-CRISPR/Cas9. (H) Representative images of FG-labeled RGCs 7 days after optic nerve crush in mice

Article Snippet: To construct adeno-associated virus (AAV) plasmids expressing Staphylococcus aureus Cas9 and single guide RNA sequences under control of the CMV and U6 promoters, respectively, doublestrand DNA oligonucleotides corresponding to the designed guide RNA sequences were inserted into a BsaI recognition site, as follows: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHAbGHpA;U6::BsaI-sgRNA (pX601; a gift from Feng Zhang; Addgene plasmid no. 61591).27 Guide RNA sequences targeting Ecel1 and cyan fluorescent protein (CFP), as a negative control, were used in this study (see Fig. 4A).

Techniques: Knockdown, CRISPR, Control, Expressing, Injection, Mutagenesis, Sequencing, Western Blot, Immunostaining, Labeling