Hero Image Ex 52 Del

cat no | io1019

ioSkeletal Myocytes
DMD Exon 52 Deletion

Human iPSC-derived Duchenne muscular dystrophy model

ioSkeletal Myocytes DMD Exon 52 Deletion are opti‑ox precision reprogrammed skeletal myocytes carrying a genetically engineered hemizygous deletion in exon 52 of the DMD gene encoding the Dystrophin protein. These cells offer a rapidly maturing, consistent, and scalable isogenic system to study Duchenne muscular dystrophy in a physiologically relevant human cell model. Use the cells to study how the exon deletion impacts muscle cell function, and investigate methods for dystrophin restoration, such as ASO-mediated exon skipping.

Place your order

Confidently investigate your phenotype of interest across multiple clones with our disease model clone panel. Detailed characterisation data (below) and bulk RNA sequencing data (upon request) help you select specific clones if required.

per vial

Benchtop benefits

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Disease-related phenotype

The disease model cells lack expression of dystrophin, as demonstrated by immunocytochemistry, making them a relevant human model for exon skipping applications.

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Consistent

Our platform ensures consistency, scalability and reproducibility, overcoming the challenges associated with the use of primary muscle cells and immortalised cell lines.

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Make True Comparisons

Pair the DMD disease model cells with the genetically matched wild-type skeletal muscle cells to study the impact of the deletion, or test methods for dystrophin restoration.

Technical data

Highly characterised and defined

ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells express skeletal muscle cell specific markers and lack expression of Dystrophin, demonstrating a Duchenne muscular dystrophy phenotype

ioSkeletal Myocytes DMD Ex 52 DMD combined ICC

Immunocytochemistry staining at day 10 post revival demonstrates robust expression of Desmin, a component of the contractile apparatus, and no expression of Dystrophin in the ioSkeletal Myocytes DMD Del Ex52/Y disease model cells, whereas ioSkeletal Myocytes, the wild type isogenic control, express both markers (upper panel). Robust expression of Myosin Heavy Chain (MHC) and the muscle transcription factor Myogenin is observed in ioSkeletal Myocytes DMD Del Ex44/Y and ioSkeletal Myocytes (lower panel). Anti-dystrophin antibody clone 2C6 (MANDYS106).

ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells demonstrate classical skeletal myocyte morphology

ioSkeletal Myocytes DMD Exon 52 classical myocyte morphology shown at day 10 post revival by bright-field imaging.

ioSkeletal Myocytes DMD Exon 52 Deletion form elongated, multinucleated myocytes over 10 days, comparable to the wild-type ioSkeletal Myocytes isogenic control. Day 3 to 10 post-revival; 100X magnification.

ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells demonstrate gene expression of key myogenic markers following reprogramming

ioSkeletal Myocytes DMD Exon 52 Deletion gene expression of key myogenic markers by RT-qPCR

Following reprogramming, ioSkeletal Myocytes DMD Exon 52 Deletion (DMD DelEx52/Y) and wild type ioSkeletal Myocytes (WT Control) downregulate expression of pluripotency genes (A), while demonstrating expected expression of key myogenic markers (B). Gene expression levels assessed by RT-qPCR. Data expressed relative to the parental human iPSC (hiPSC), normalised to HMBS. Data represents day 10 post-revival samples.

Disease-related phenotype

ioSkeletal Myocytes DMD Exon Deletion disease model cells show absence of Dystrophin protein by immunocytochemistry, demonstrating a Duchenne muscular dystrophy phenotype

 ioSkeletal Myocytes DMD Exon Deletion disease model cells immunocytochemistry shows absence of Dystrophin protein

ioSkeletal Myocytes DMD Exon 44 Deletion and DMD Exon 52 Deletion disease model cells, and ioSkeletal Myocytes wild type isogenic control, were cultured in 96-well plates at a density of 32,000 cells per well, according to the user manual. Immunocytochemistry staining for Dystrophin and Myosin Heavy Chain (MHC) was carried out at day 10 post-revival. The data show robust expression of MHC, but no expression of Dystrophin in the ioSkeletal Myocytes DMD Del Ex44/Y or DMD Del Ex52/Y disease model cells compared to the wild-type control, demonstrating a Duchenne muscular dystrophy phenotype. Data courtesy of Charles River Laboratories.

ioSkeletal Myocytes DMD Deletion disease model cells are amenable to ASO-mediated Dystrophin restoration, demonstrated with the DMD Exon 44 Deletion product

ioSkeletal Myocytes DMD Exon 52 Deletion disease model cells show dystrophin restoration by ASO-mediated exon skipping

ioSkeletal Myocytes (wild type control) and ioSkeletal Myocytes DMD Exon 44 Deletion (DMD Del Ex44) were cultured according to the user manual in 96-well plates at a density of 32,000 cells per well. On day 4 post-revival the cells were treated by gymnosis with exon 45 skipping antisense oligonucleotide (ASO-1), concentration range 1-50 µM for immunocytochemistry and high content image (HCI) analysis, and 0.01-50 µM for ddPCR. Cells were cultured to day 7 then analysed.

A) Dystrophin mRNA restoration: RNA was extracted and cDNA synthesised. PCR primers and a fluorescent (FAM) labelled probe were designed to amplify the region coding exons 43-45 (non-skip, NSKP) or exons 43-46 (skip, SKP). PCR was carried out to quantify the non-skip and skip transcripts. The graph shows a concentration-dependent increase in the amount of SKP transcript (blue) and a decrease in the NSKP transcript (yellow), indicating that ASO-1 treatment has been successful in creating an in frame mRNA transcript for dystrophin.
B) Dystrophin protein restoration: cells were stained for dystrophin, myosin heavy chain (not shown) and DAPI for cell nuclei quantification (not shown) and measured by high content analysis of images captured on Yokogawa Cell Voyager 8000 using a proprietary algorithm. The graph shows dystrophin restoration levels calculated as % of dystrophin/MHC area compared to wild type. ASO-1 treatment increased dystrophin protein expression in a concentration-dependent manner.

Each condition was tested in technical duplicate for ddPCR and technical triplicate for HCI analysis, and in biological duplicate (N=2). Data courtesy of Charles River Laboratories.

Seeding density

Recommended seeding density for ioSkeletal Myocytes DMD Del Ex52/Y is 100,000 cells/cm2, matching the wild type control

ioSkeletal Myocytes DMD Del Ex52/Y are compatible with plates ranging from 6 to 384 wells.
The recommended seeding density is 100,000 cells/cm2. One small vial can plate a minimum of 0.5 x 24-well plate, 0.75 x 96-well plate, or 1 x 384-well plate. 

Cells arrive ready to plate

Timeline for culturing bit.bio iPSC-derived skeletal myocytes

ioSkeletal Myocytes DMD Exon 52 Deletion are delivered in a cryopreserved format and are programmed to mature rapidly upon revival in the recommended medium. The protocol for the generation of these cells is a two-phase process: Phase 1. Stabilisation for 3 days. Phase 2. Maintenance during which the skeletal myocytes mature.

Product information

Starting material

Human iPSC line

Karyotype

Normal (46, XY)

Seeding compatibility

6, 12, 24, 48, 96 & 384 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >2.5 x 106 viable cells
Large: >5 x 106 viable cells

Quality control

Sterility, protein expression (ICC), gene expression (RT-qPCR) and genotype validation (Sanger sequencing)

Differentiation method

opti-ox cellular reprogramming

Recommended seeding density

100,000 cells/cm2

User storage

LN2 or -150°C

Format

Cryopreserved cells

Genetic modification

Hemizygous exon 52 deletion in the DMD gene

Applications

ASO-mediated exon skipping
Muscular dystrophy research
Dystrophin restoration
Muscle disease modelling

Product use

ioCells are for research use only

Product resources

ioSkeletal Myocytes Brochure
ioSkeletal Myocytes

bit.bio

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Read this blog on skeletal myocytes cell culture for our top tips on careful handling, cell plating and media changes to achieve success from the outset.

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