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photoreceptor derived 661w cell line  (ATCC)
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
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blue light photoreceptor  (Makita)
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
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photoreceptor reporter system  (Addgene inc)
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in <t>661W</t> cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.
Photoreceptor Reporter System, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in 661W cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.

Journal: Molecular Vision

Article Title: RNA interference to reduce Egr1 expression in rods delays retinal degeneration in a model of retinitis pigmentosa

doi:

Figure Lengend Snippet: In vitro validation of shEgr1 and mEgr1 sequences and design of AAV vectors. A : Real time PCR for Egr1 in 661W cell lines stably expressing shEgr1. Each cell line (plotted on the x-axis) expresses a different shEgr1 sequence (1–6, ). Sequence (1) showed the strongest downregulation (78%, dotted line) and was chosen for further tests. Shown are individual data points and means ± SD n=6. One-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. B : PMA stress test in 661W wt and 661W-shEgr1 (1) cells. Shown are individual data points and means ± SD n=7. Two-way ANOVA with Dunnett’s multiple comparisons test, p values as indicated. C : In vitro test of the Egr1 overexpression plasmid AAV2::CMV-mEgr1-P2A-eGFP-pA-WPRE (CMV-mEgr1) in 661W cells. Egr1 mRNA levels were determined at 24 h and 48 h after transfection of the overexpressing plasmid (pink) and compared to cells that were not transfected (blue) and cells that received the pcDNA3-CMV-eGFP control plasmid (purple). Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. D : Expression levels of the EGR1 target gene Atf3 in untransfected (blue), ctrl-transfected (purple) and CMV-mEgr1-transfected (pink) cells at 48 h after transfection. Shown are individual data points and means ± SD n=3. One-way ANOVA with Tukey’s multiple comparisons test, p values as indicated. E : Schematic representations of the Egr1 downregulation (AAV2- shEgr1 , top), Egr1 overexpression (AAV2- mEgr1 , middle), and control (AAV2-ctrl, bottom) AAV vectors. ITR2: inverted terminal repeats of serotype 2; mOP: mouse opsin promoter (rod-specific); OPN1MW : human opsin medium wavelength promoter (cone-specific); (e)GFP: (enhanced) green fluorescent protein; pA: poly-A tail; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element; miR-E: microRNA-E backbone for shRNAs; P2A: 2A self-cleaving peptide.

Article Snippet: The photoreceptor-derived 661W cell line [ ] and HEK293T (ATCC CRL-3216) cells were cultured in DMEM, 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific, Waltham, MA) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific) at 37 °C and 5% CO 2 .

Techniques: In Vitro, Biomarker Discovery, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Sequencing, Over Expression, Plasmid Preparation, Transfection, Control, Virus