Journal: Molecular Therapy. Nucleic Acids
Article Title: Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing
Figure Lengend Snippet: Detection of 5′ trans-splicing in vitro . (a) Left: Anti-Flag Western Blot on total proteins from 3T3 cells transfected with 5′-PTM or a plasmid expressing full length Dnm2-Flag fusion protein as control (Dynaflag). NT: nontransfected. Right: Dnm2 immunoprecipitation followed by Flag western blot on 3T3 cells transfected with 5′-PTM. The image is overexposed and over contrasted to enhance the detection of the band indicated by an asterisk. (b) RT-PCR analysis on mRNA extracted from 3T3 cells transfected with 5′-PTM. Dyna-endo: endogenous Dnm2 transcripts amplified using E10EndoF/E12endoR primers, TS: trans-spliced RNA amplified using E8OptiF/Ex17endoR and Ex11OptiF/Ex14endoR primers; PTM are amplified using E8OptF/Ex11OptiR primers. C−: negative PCR control. The isoform of Dnm2 identified after sequencing for each band is indicated below ( i.e. , isoform 2 or 4 or both). 3T3 cells were transfected three times. (c) Top: Schematic representation of Dnm2 alternative splicing leading to isoforms 2 and 4, locations of primers used for PCR amplification are depicted. Bottom: Example of heterozygous trans-spliced sequence showing Ex13 optimized sequence followed by ex13 bis and ex14 endogenous sequence. The orange squares show the optimized nucleotides. (d) Anti-Flag and anti-DNM2 immunofluorescence on 3T3 cells 48 hours after transfection with AS2-, AS3-, noAS-▵pA-5′-PTM and full length Dnm2-Flag fusion constructs (scale bar = 10 µm). PTM, pre-trans-splicing molecules; RT-PCR, reverse transcription-polymerase chain reaction.
Article Snippet: Total RNA (1 µg) was submitted to reverse transcription using the Superscript III reverse transcriptase kit (Life Technologies) using random primers. cDNA were amplified by PCR under the following conditions: 96°C for 5 minutes, cycles of 30 seconds at 96°C, 30 seconds at the appropriate temperature (58 to 61°C), 30 seconds to 1 minute at 72°C, and a final step of 7 minutes at 72°C.
Techniques: In Vitro, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Sequencing, Immunofluorescence, Construct