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1) Product Images from "Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina"
Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina
Figure Legend Snippet: Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p
Techniques Used: CRISPR, Injection, Two Tailed Test
Figure Legend Snippet: In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.
Techniques Used: In Vitro, CRISPR, Construct, Plasmid Preparation, Western Blot, Expressing, Transfection, FACS
Figure Legend Snippet: Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p
Techniques Used: CRISPR, In Vivo, Expressing, Cell Counting
Figure Legend Snippet: Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.
Techniques Used: Agarose Gel Electrophoresis, Western Blot, In Situ, In Vitro, Expressing, CRISPR, Construct, Stripping Membranes
Figure Legend Snippet: Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p
Techniques Used: Cell Counting
Figure Legend Snippet: Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.
Techniques Used: CRISPR, Plasmid Preparation