B6-hDMD (E49-53) Mice

Catalog Number: I001133

Strain Name: C57BL/6NCya-Dmdtm3(hDMD Exon 49-53)/Cya

Genetic Background: C57BL/6NCya

One of Cyagen's HUGO-GT™ (Humanized Genomic Ortholog for Gene Therapy) Strains

 

Strain Description

Duchenne Muscular Dystrophy (DMD) is a severe, progressive, and disabling X-linked recessive genetic disorder characterized primarily by muscle atrophy. This disease leads to motor impairments, eventually requiring assisted ventilation, and often results in premature death. The primary cause of DMD is mutations in the DMD gene, which encodes the dystrophin protein. These mutations lead to a reduction or absence of dystrophin in muscle tissue, resulting in muscle atrophy and related complications [1]. The lack of dystrophin leads to the breakdown of the dystrophin-associated protein complex (DAPC) within the muscle membrane, disrupting the interaction between actin and the extracellular matrix, making the muscles more susceptible to damage. This susceptibility results in the gradual loss of muscle tissue and function, potentially leading to cardiomyopathy [2]. Researchers have identified thousands of different DMD gene mutations in patients with DMD. Deletion mutations account for approximately 60%–70%, while duplication mutations account for 5%–15%. These mutations are primarily concentrated in hotspot regions of the DMD gene, specifically between exons 45-55 (47%) and exons 3-9 (7%) [1].

Currently, gene therapy approaches for Duchenne Muscular Dystrophy (DMD) primarily include exon skipping and AAV supplementation, as well as emerging gene editing techniques like CRISPR. The exon skipping strategy involves using antisense oligonucleotide (ASO) drugs to bind to specific sequences of pre-mRNA, skipping the mutated exon and restoring the open reading frame (ORF) integrity, thus producing a truncated but partially functional dystrophin protein. Several ASO drugs targeting the DMD gene have been approved, such as Eteplirsen (targeting exon 51), Golodirsen (targeting exon 53), and Casimersen (targeting exon 45) developed by Sarepta, and Viltolarsen (targeting exon 53) developed by Nippon Shinyaku. Since most ASO and CRISPR-based gene editing therapies target the human DMD gene, humanizing mouse genes helps accelerate clinical applications for DMD therapies, considering the genetic differences between animals and humans.

The B6-hDMD (E49-53) mouse is a humanized model of exons 49-53 of the Dmd gene, used for researching Duchenne Muscular Dystrophy. Homozygotes are viable and fertile. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen provides other humanized models such as [hE49-53, del E50], [hE44-45], [hE44-45, del E44], [hE44-45, c.6438+2 T to A], and [hE8-30], covering most popular research areas and offering customized services based on different mutation needs.

Strain Strategy


Figure 1. Gene editing strategy of B6-hDMD (E49-53) mice. The partial intron 48 (~5kb) to partial intron 53 (~5kb) of mouse Dmd will be replaced with the partial intron 48 (~5kb) to partial intron 53 (~5kb) of human DMD.

Application

  • Research on the pathogenesis of Duchenne Muscular Dystrophy (DMD);
  • Preclinical efficacy evaluation of DMD therapeutic drugs.

 

Validation Data

1. RT-PCR

Figure 2. RT-PCR detection of gene expression in B6-hDMD (E49-53) mice. cDNA was obtained by reverse transcription using primers specifically paired with human DMD mRNA, followed by gel electrophoresis to determine the expression of the human DMD gene. The results show the presence of human DMD cDNA bands in B6-hDMD (E49-53) mouse tissues, with band sizes matching the expected values.

Note: Due to the length of the target band sequence, two pairs of primers were designed for amplification; the first four bands are amplified by the F1/R1 primer pair, and the latter four bands are amplified by the F2/R2 primer pair.

2. cDNA sequencing

Figure 3. Gene sequencing results of the exon 49-53 region in B6-hDMD (E49-53) mice. Sequencing analysis of the DMD cDNA reverse-transcribed from mRNA shows that the exon 49-53 region of the Dmd gene in B6-hDMD (E49-53) mice corresponds to the respective sequences in the human DMD gene, with nucleotide sequences identical to the reference sequences in the human DMD gene.

3. Gene expression

Figure 4. RT-qPCR Analysis in 6-week-old male homozygous B6-hDMD(E49-53) and Wild-Type (WT) Mice. The RT-qPCR results show that the human DMD gene is significantly expressed in the skeletal muscle, cerebral cortex, and heart of B6-hDMD(E49-53) mice, while no expression is detected in wild-type mice. The mouse Dmd gene is significantly expressed in wild-type mice but is not expressed in B6-hDMD(E49-53) mice. (Bars represent mean ± SEM，n=4)

Note: The primers that detect the expression of the human DMD gene and the mouse Dmd gene target the Exon 50-51 region.

4. Protein expression

Figure 5. Western blot analysis of DMD protein expression in 6-week-old male homozygous B6-hDMD(E49-53) and Wild-Type (WT) Mice. The Western Blot analysis shows that B6-hDMD(E49-53) mice normally express the DMD protein in skeletal muscle, cerebral cortex, and heart.

Note: The antibody used for detecting mouse DMD protein targets aa. 3550-3678. The gene sequence encoding this binding region is located after the humanized sequence (approximately in the exon 78-79 region).

5. Serum Creatine Kinase (CK) Level

Figure 6. Serum creatine kinase (CK) levels in 6-week-old male homozygous B6-hDMD(E49-53) and wild-type (WT) mice. Creatine kinase is a component of the myocardial enzyme spectrum, and elevated serum creatine kinase levels are related to muscle and myocardial damage. The blood biochemical results show that the CK values in B6-hDMD(E49-53) mice are similar to those in wild-type mice. (Bars represent mean ± SEM, n=4)


 

References

[1] Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy. Nat Rev Dis Primers. 2021 Feb 18;7(1):13.

[2] Babbs A, Chatzopoulou M, Edwards B, Squire SE, Wilkinson IVL, Wynne GM, Russell AJ, Davies KE. From diagnosis to therapy in Duchenne muscular dystrophy. Biochem Soc Trans. 2020 Jun 30;48(3):813-821.