Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System

Abstract

Background: Dystrophin Dp71 is one of the isoforms produced by the DMD gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. Methods: Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMDmdx mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. Results: Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression. Conclusions: Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.

Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice

“…The reproducibility of dystrophin quantification by Wes was in line with the FDA/EMA regulations for quantitative biochemical analysis [10]. Since it is a relatively new method, so far only a few studies have been published making use of this methodology [10,50]. In this study, we applied Wes, allowing accurate quantification of the amount of dystrophin produced by exon 23 skipping in the mdx mouse.…”

“…The produced chemiluminescent signal is automatically quantified and is displayed as an electropherogram showing automatically detected intensity peaks that can be quantified by calculation of the area under the curve. Besides quantification of the fulllength Dp427 dystrophin isoform, Wes can also be used to quantify levels of shorter dystrophin isoforms, such as, for example, the Dp71 isoform [10,50]. We validated this method by assessing linearity of quantification for different antibodies, reproducibility (interassay and intra-assay, interoperator), and found the assay to be highly sensitive and linear over a wide dynamic range and with a lower limit of quantification (LLOQ) low enough to quantify trace dystrophin levels present in skeletal muscle of DMD patients.…”

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

“…The deltaE50-MD dog model of DMD exhibits markedly more severe muscle pathological changes than adult mdx mice, with widespread areas of focal necrosis and regeneration even at 15 months of age, thus increased labelling of dp71 is not unexpected (marked increases in dp71 expression have also been reported in muscle of young mdx mice, i.e. in the acute phase of disease onset 61 ).…”

Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells

“…. Plasmids expressing Dp71 and Dp71ab were constructed by inserting the coding sequences of Dp71 and Dp71ab, respectively (Supplementary Table 1), into the mammalian expression vector pcDNA3 with a CMV promoter (Invitrogen, Thermo Fisher Scientific Inc.) by FASMAC Co., Ltd, as described 40 . AGS cells (4 × 10 5 ) grown to 80% confluency on six-well culture dishes were transfected with 2 μg of the synthesized plasmids in 3 μL Lipofectamine 3000 (Thermo Fischer Scientific).…”