Journal: bioRxiv

Article Title: CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

doi: 10.1101/2023.04.18.536394

Figure Lengend Snippet: (A) Scheme of a dual reporter plasmids. To study the impact of several modifications of the 3’UTR of utrophin, several 3’UTR variants were integrated after the Gaussia luciferase (Gluc) gene, driven by an SV40 promoter. The secreted alkaline phosphatase (SEAP) expression, under the control of a CMV promoter, was used for transfection efficacy and normalization. miR BS and stability, localisation and AU-Rich element predictions are indicated with their nucleotide positions. (B) The reporter constructs were transfected in hDMD myoblasts and Gluc and SeAP expression measured 48hrs post-transfection. Left; 3’UTR variants used in this study. For each variant it is indicated the nucleotide position of the deletion. Right: ratio of Gluc/SEAP was calculated for each contract. The reporter construct 1 (Full length) was used to determine the basal level of Gluc expression and construct 12 (Full length reverse) as negative control. Bars represent mean ± SEM of n = 3 per condition. Dots represent each experiment. *p < 0.05, **p < 0.01, ***p < 0.001 versus full length; +p < 0.05, ++p < 0.01, +++p < 0.001 versus D341-2046 3’UTR variant.

Article Snippet: All utrophin 3’UTR reporter constructs were generated by GenScript Biotech (Leiden, Netherlands) and inserted in the pEZX-GA02 Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) reporter cloning vector (ZX-104, Genecopoeia) downstream of the Gaussia luciferase reporter gene.

Techniques: Luciferase, Expressing, Control, Transfection, Construct, Variant Assay, Negative Control