Journal: Nature medicine
Article Title: A novel DMD IRES results in a functional N-truncated dystrophin, providing a potential route to therapy for patients with 5’ mutations
doi: 10.1038/nm.3628
Figure Lengend Snippet: (a) Induction of translation of the downstream (FLuc) cistron in an in vitro transcription/translation system (rabbit reticulocyte lysate [RRL], left) and following transfection into C2C12 cells (right) in a dicistronic dual luciferase reporter. Results are expressed as the ratio of Firefly:Renilla luciferase (F/L), and normalized to the empty vector (set as 1). (b) Formaldehyde electrophoresis of the T7 transcription products used in the RRL assay. (c) Dicistronic mapping constructs (left) used to map cap-independent translation activity. FLuc luminescence (cap-independent) is expressed as a percentage of RLuc luminescence (cap-dependent) after transfection in C2C12 cells (right). All results were normalized to the exon 6 alone vector, the FLuc:RLuc ratio of which was set at a value of 1. Statistical analysis was performed using a Kruskal-Wallis test, comparing the results for each construct versus the exon 6 alone vector. Exon 1 to 6 (p<.0001), exon 2 to 6 (p=0.0175), exon 3 to 6 (p=0.0009), exon 3* to 6 (p=0.0078), exon 4 to 6 (p=0.0078), and exon 5 to 6 (p=0.0019). Deletion of exon 5 (either in whole or in part): no significant difference in Fluc translation in comparison to exon 6 alone. (d) RT-PCR products amplified from RNA derived from transfected C2C12 cells, using primers located as depicted as arrows on the scheme in panel (c). (e) Northern blot analysis of C2C12 transfected cells using a P 32 radiolabeled probe targeting the FLuc. (A non-specific band of approximately 3 kb is detected in every transfection condition, including the empty vector, and is therefore unrelated to the increase in FLuc or EMCV signals compared to empty vector.) (f) IRES activity in the presence of either a duplicated or a deleted exon 2. Error bars represent s.d.
Article Snippet: All primers used to synthesis reporter constructs are available upon request. eMCV (Encephalomyocarditis virus) IRES sequence was amplified from pWPI (Addgene).
Techniques: In Vitro, Transfection, Luciferase, Plasmid Preparation, Electrophoresis, Construct, Activity Assay, Comparison, Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Northern Blot
Addgene inc is a verified supplier 
![(a) Induction of translation of the downstream (FLuc) cistron in an in vitro transcription/translation system (rabbit reticulocyte lysate [RRL], left) and following transfection into C2C12 cells (right) in a dicistronic dual luciferase reporter. Results are expressed as the ratio of Firefly:Renilla luciferase (F/L), and normalized to the empty vector (set as 1). (b) Formaldehyde electrophoresis of the T7 transcription products used in the RRL assay. (c) Dicistronic mapping constructs (left) used to map cap-independent translation activity. FLuc luminescence (cap-independent) is expressed as a percentage of RLuc luminescence (cap-dependent) after transfection in C2C12 cells (right). All results were normalized to the exon 6 alone vector, the FLuc:RLuc ratio of which was set at a value of 1. Statistical analysis was performed using a Kruskal-Wallis test, comparing the results for each construct versus the exon 6 alone vector. Exon 1 to 6 (p<.0001), exon 2 to 6 (p=0.0175), exon 3 to 6 (p=0.0009), exon 3* to 6 (p=0.0078), exon 4 to 6 (p=0.0078), and exon 5 to 6 (p=0.0019). Deletion of exon 5 (either in whole or in part): no significant difference in Fluc translation in comparison to exon 6 alone. (d) RT-PCR products amplified from RNA derived from transfected C2C12 cells, using primers located as depicted as arrows on the scheme in panel (c). (e) Northern blot analysis of C2C12 transfected cells using a P 32 radiolabeled probe targeting the FLuc. (A non-specific band of approximately 3 kb is detected in every transfection condition, including the empty vector, and is therefore unrelated to the increase in FLuc or <t>EMCV</t> signals compared to empty vector.) (f) <t>IRES</t> activity in the presence of either a duplicated or a deleted exon 2. Error bars represent s.d.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5597/pmc04165597/pmc04165597__nihms602834f2.jpg)