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Results: 5

1.
Figure 5

Figure 5. From: RNA Interference Targeting CUG Repeats in a Mouse Model of Myotonic Dystrophy.

Duration of siCAG response. (a) Slot-blot hybridization assay to determine the levels of CUGexp accumulation in tibialis anterior muscle. Muscle was harvested 7 or 14 days after electroporating 3 µg of siCAG. Hybridization signal from (CAG)8 probe (CUGexp RNA) was normalized to mouse skeletal actin probe. Each bar represents the mean ± SD for indicated number of mice and bases on two independent hybridization experiments. *P < 0.0001, **P < 0.05 for siRNA versus untreated muscle (Student's t-test). (b) RT-PCR splicing assay for five MBNL1-dependent exons. Analyses were performed 7 or 14 days after electroporating 3 µg of siCAG (si) or saline (sal).

Krzysztof Sobczak, et al. Mol Ther. 2013 February;21(2):380-387.
2.
Figure 2

Figure 2. From: RNA Interference Targeting CUG Repeats in a Mouse Model of Myotonic Dystrophy.

Nuclear foci of CUGexp RNA and MBNL1 protein are dispersed in muscles treated with siCAG. (a) Fluorescence in situ hybridization (FISH) shows reduction of CUGexp foci (red) in nuclei (blue) in isolated flexor digitorum brevis (FDB) muscle fibers. Muscle was taken 7 days after electroporation of siCAG or saline. Inset shows magnification of selected nuclei. Nuclei are counterstained blue using 4,6 diamino-2-phenylindole dihydrochloride (DAPI). (b) Combined FISH for CUGexp RNA (red) and immunofluorescence for MBNL1 protein (green) in transverse sections of tibialis anterior muscle from human skeletal actin—long repeat mice. Muscle was taken 7 days after electroporation of siCAG or saline. The CUGexp foci are dispersed and the MBNL1 distribution is more diffuse in siCAG-treated muscle. Nuclei are counterstained blue (DAPI). (c) Immunofluorescence of isolated FDB muscle fibers shows redistribution of MBNL1 (green) from punctate to diffuse localization in the nucleus (blue), 7 days after electroporation of siCAG.Scale bars = 5 µm. Objective: 100× Plan Apo, 1.4 NA oil.

Krzysztof Sobczak, et al. Mol Ther. 2013 February;21(2):380-387.
3.
Figure 1

Figure 1. From: RNA Interference Targeting CUG Repeats in a Mouse Model of Myotonic Dystrophy.

siRNA directed against CUG repeats (siCAG) causes knockdown of CUGexp expression and reduction of nuclear RNA foci in mouse skeletal muscle in vivo. (a) Sequences of siRNAs used in this study; gray box shows the putative siRNA nuclear localization signal in the siCAG-tail duplex. (b) Northern blot for CUGexp transcript in RNA from tibialis anterior (TA) muscle. RNA was isolated 7 days after electroporation of siCAG or siCAG tail (si), as compared with saline-electroporation control (sal). Results were normalized to endogenous mouse Acta1 for quantification on graph (mean ± SD, at least four independent experiments, * denotes P < 0.0001, t-test). (c) qRT-PCR for endogenous transcripts containing CUG repeats (Mapkap1, Txlnb, and Tnfrsf22) or CAG repeats (Nr3c1 and Dap). The number of repeats in each transcript is indicated. TA muscle from human skeletal actin—long repeat (HSALR) mice was taken 7 days after electroporation of saline, siCAG or siCAG tail (n = 3 mice per each group, *P < 0.0001, t-test). Expression was normalized to Gtf2b, a housekeeping transcript that shows low variance in wild type or HSALR muscle.12 WT, wild type.

Krzysztof Sobczak, et al. Mol Ther. 2013 February;21(2):380-387.
4.
Figure 4

Figure 4. From: RNA Interference Targeting CUG Repeats in a Mouse Model of Myotonic Dystrophy.

Dose response relationship for siCAG. (a) The level of CUGexp transcript was determined by northern analysis in tibialis anterior (TA) muscle. Four doses of siCAG (3, 1, 0.3, or 0.1 µg) were electroporated into TA of human skeletal actin—long repeat mice (n = 2 per each group). Total RNA was isolated after 7 days. Saline electroporated TA serves as control. (b) RT-PCR analysis of alternative splicing for mice described in (a). The two higher siCAG doses produced strong splicing correction, while the lowest dose had no effect. (c) Reduction of myotonia 1 week after electroporation of siCAG into TA muscle. The contralateral TA was electroporated with saline alone (n = 2 mice for each siRNA dose; *P < 0.0001 for siCAG versus saline according to Student's t-test). Myotonic discharges were rated by a blinded examiner using extracellular needle electrode recordings (electromyography) under general anesthesia. Myotonia was significantly reduced in muscles treated with 3 and 1 µg of siCAG. HSALR, human skeletal actin—long repeat mouse; WT, wild type mouse. **P < 0.05.

Krzysztof Sobczak, et al. Mol Ther. 2013 February;21(2):380-387.
5.
Figure 3

Figure 3. From: RNA Interference Targeting CUG Repeats in a Mouse Model of Myotonic Dystrophy.

Effect of siCAG and siCAG tail on MBNL1 splicing regulatory activity. (a) RT-PCR analysis of alternative splicing for exon 22 (ex.22) of Atp2a1, exon 8 (ex.8) of Arfgap2, and exon 13 (ex.13) of Camk2b, three exons whose splicing is promoted by MBNL1. Splice products in wild-type mice are shown on the right side of gel. In response to siCAG, partial correction of alternative splicing was observed for each exon. RNA was extracted from tibialis anterior (TA) muscle of human skeletal actin—long repeat mice, 7 days after electroporation of siCAG or siCAG tail (si). The contralateral TA was electroporated with saline (sal). (b) Same as in (b), except that RT-PCR analysis was performed on exons whose alternative splicing is normally repressed by MBNL1: M-line region exon 5 (Mex.5) of Titin, exon 7 (ex.7) of Nfix, and exon 11 (ex.11) of Ldb3. (c) Quantification of results in (b) and (c), based on three independent experiments (mean ± SD). Results are expressed as the percentage of splice products including the alternative exons for four groups of animals (*P < 0.0001, **P < 0.05, Student's t-test for comparison with saline-treated muscles). HSALR, human skeletal actin—long repeat mouse; WT, wild type mouse.

Krzysztof Sobczak, et al. Mol Ther. 2013 February;21(2):380-387.

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