Home › Cell Biology › Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy
Cell Biology JoVE (Open Access) Citable · DOI

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

DOI: 10.3791/53776-v
What you'll learn
  • ✓ Design and execute multi-exon skipping antisense oligonucleotide cocktails targeting DMD genes
  • ✓ Transfect dog myoblasts with 2'OMePS and morpholino oligonucleotides for exon-skipping validation
  • ✓ Perform intramuscular and systemic injections in canine DMD models to assess dystrophin rescue
  • ✓ Evaluate multi-exon skipping efficacy using RNA extraction and molecular analysis
Protocol

Exon skipping is currently a most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

Difficulty
advanced
Total time
~4–6 weeks (including transfection optimization, injection recovery, and tissue analysis)
Model organism
Dog (Canis lupus familiaris), X-linked muscular dystrophy model
Biosafety
BSL-1

Steps

1
Transfect dog myoblasts with 2'OMePS oligonucleotides

Introduce 2'O-methyl phosphorothioate antisense oligonucleotides into canine myoblast cultures to induce exon skipping of dystrophin gene targets. Optimize transfection conditions and confirm cellular uptake.

▶ 01:23
2
Transfect myoblasts with morpholino and extract RNA

Transfect dog myoblasts with morpholino oligonucleotides targeting DMD exons. Extract total RNA from treated and control cells to assess splicing patterns and exon-skipping efficiency.

▶ 02:59
3
Perform intramuscular injections and muscle biopsies

Administer cocktail antisense oligonucleotides via intramuscular injection into diseased dog muscle or obtain open muscle biopsies. Collect tissue samples for downstream molecular and histological analysis.

▶ 05:18
4
Administer systemic antisense oligonucleotide injections

Deliver multi-exon skipping cocktail via systemic injection routes (intravenous or intraperitoneal) to achieve whole-body dystrophin rescue in the canine DMD model.

▶ 06:42
5
Analyze multi-exon skipping and dystrophin expression

Evaluate exon-skipping patterns in treated tissues using RT-PCR, RNA-seq, or similar methods. Assess dystrophin protein expression and localization via Western blot and immunofluorescence to quantify therapeutic rescue.

▶ 07:39
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