Table 4.
Details of RNA imaging experiment design used in studies of CUG and CGG repeat toxicity in DM1 and FXTAS
| CUG-repeat transcript imaging | CGG-repeat transcript imaging | |
|---|---|---|
| Goals | • movement of mutant transcripts • dynamics of foci formation • RNA co-localization with Mbnl1 • Mbnl1 role in foci formation |
• RNA foci formation and stability • foci dynamics during the cell cycle • effect of drugs on foci formation • RNA co-localization with Sam68 |
| System | MS2 | Spinach2 |
| Number of aptamers | 24 | 1 |
| Localization of aptamers | Upstream of 3’UTR | Upstream of the polyA signal |
| Fluorescent protein/dye | GFP/mCherry | DFHBI |
| Delivery method | Retroviral vectors | Plasmid vectors |
| Promoter | TRE (inducible) | CMV (strong, non-inducible) |
| Additional techniques | FRAP, FLIP, imaged simultaneously with protein | imaged simultaneously with protein |
| Cell type | C2C12 myoblasts | COS-7 |
| Temporal resolution | Every 333 msec for 20 sec or every 30 sec for up to 23 min |
Every 20 min for 6 h. |
| Microscopy | Spinning disk confocal microscopy | Epifluorescence microscopy |
| Software | MetaMorph software | NIS-Elements software |
| Comment | • Higher temporal resolution is needed for the analysis of transcript movement. • Standard microscopic filters can be used. • Protein-RNA systems enable photobleaching. • Single RNA granule tracking is possible with 24 MS2 hairpins |
• Longer observation is needed for changes during the cell cycle. • Fast dye exchange reduces photobleaching. • DFHBI exhibits better fluorescence signal stability than GFP. • DFHBI can be replaced with DFHBI-1T for standard microscopic filters |
| Reference | Querido et al. 2011 | Strack et al. 2013 |