Name: huABCA4-c.5461-10T>C Mouse
Brand: サイヤジェン（Cyagen）
SKU: C001966

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huABCA4-c.5461-10T>C Mouse

製品名

huABCA4-c.5461-10T>C Mouse

製品ID

C001966

系統名

C57BL/6JCya-Abca4^{tm1(hABCA4*c.5461-10T>C)}/Cya

背景情報

C57BL/6JCya

Note

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

状況

このマウス系統を論文で使用する場合は、「huABCA4-c.5461-10T>C Mouse（カタログ番号C001966）はサイアジェンから購入しました。」と引用してください。

HUGO-GT Humanized Models

Disease Animal Models

Small Nucleic Acids

製品タイプ

年齢

遺伝子型

性別

数量

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HUGO-GT Humanized Models

Disease Animal Models

Small Nucleic Acids

基本情報

検証 Data

系統詳細

Stargardt Disease (STGD) is a hereditary macular dystrophy marked by yellowish fusiform spots in the retinal pigment epithelium, leading to macular atrophy. It primarily affects children and adolescents, causing progressive central vision loss and mild color vision impairment. The fundus may show pale yellow lesions with gold foil-like reflections and yellow-white spots around the posterior pole. Advanced stages involve atrophy of the retinal pigment epithelium, photoreceptor cells, and choriocapillaris. STGD is also common in sporadic cases and more frequent in children of consanguineous marriages. It affects both eyes bilaterally and progresses synchronously without significant gender differences, with an incidence of approximately 1/8000 to 1/13000. STGD is an autosomal recessive retinal disease caused by ABCA4 gene mutations, accounting for 95% of cases.

The ABCA4 gene encodes a retina-specific ABC transporter protein that removes retinal derivatives and toxic metabolites after rhodopsin photobleaching. Mutations in ABCA4 lead to the accumulation of these substances, causing apoptosis of retinal pigment epithelial and photoreceptor cells, resulting in retinal degenerative diseases. ABCA4 mutations are linked to Stargardt Disease (STGD), Cone-rod Dystrophy (CRD), and Retinitis Pigmentosa (RP). The clinical phenotype depends on the extent of ABCA4 mutations, with severe and mild mutations or two moderate mutations predisposing to STGD, and one moderate mutation predisposing to CRD^[2-4].

Currently, the drug pipeline for treating Stargardt disease (STGD) primarily focuses on supplemental delivery methods for ABCA4-targeted drugs. Among them, ProQR has developed a therapeutic antisense oligonucleotide (ASO) drug, QR-1011, which targets the c.5461-10T>C mutation ^[1]. Most ASO medicines and gene therapies act on the human ABCA4 gene. Considering the genetic differences between animals and humans, modifying mouse genes to be more human-like would help accelerate gene therapies targeting ABCA4 into the clinical stage.

The huABCA4-c.5461-10T>C mouse is a humanized model of the Abca4 gene, where the mouse Abca4 gene has been replaced with the human ABCA4 gene carrying the c.5461-10T>C mutation using gene editing technology. This model can be used for research on various retinal degeneration diseases such as Stargardt disease (STGD), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also provide customized services for specific mutations to meet the experimental needs in pharmacology and other fields.

参考文献

Kaltak M, de Bruijn P, Piccolo D, et al. Antisense oligonucleotide therapy corrects splicing in the common Stargardt disease type 1-causing variant ABCA4 c.5461-10T>C. Mol Ther Nucleic Acids. 2023 Feb 18;31:674-688.

Roberts L J , Nossek C A , Greenberg L J ,et al.Stargardt macular dystrophy: common ABCA4 mutations in South Africa—establishment of a rapid genetic test and relating risk to patients[J].Molecular Vision, 2012, 18(31-33):280-289.

Aukrust, IngvildJansson, Ragnhild W.Bredrup, CecilieRusaas, Hilde E.Berland, SirenJorgensen, AgneteHaug, Marte G.Rodahl, EyvindHouge, GunnarKnappskog, Per M.The intronic ABCA4 c.5461-10T > C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA4 protein level[J]. Acta ophthalmologica, 2017.

[4]ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease[J]. Genome Research, 2018.

系統作製戦略

The sequences from the start codon to the stop codon of the mouse Abca4 gene will be replaced with the sequences from the start codon to the stop codon of the human ABCA4 gene. The c.5461-10 T to C point mutation was introduced into intron 38 of human ABCA4.

Figure 1. Gene editing strategy of huABCA4-c.5461-10T>C mice.

適用分野

Research on Stargardt Disease (STGD);

Research on Cone-rod Dystrophy (CRD);

Research on Retinitis Pigmentosa (RP).

検証 Data

1. Expression of human ABCA4 and mouse Abca4 genes

RT-qPCR result shows a significant expression of the human ABCA4 gene in the eyes of huABCA4 mice and huABCA4-c.5461-10T>C mice carrying the point mutation. The humanized models do not express the mouse Abca4 gene, while only the mouse Abca4 gene is expressed in wild-type mice.

Figure 2. Detection of human ABCA4 and mouse Abca4 gene expression in the eyes of 4-week-old female huABCA4-c.5461-10T>C mice, huABCA4 mice, and wild-type (WT) mice (ND: Not detected).
*The c.5461-10T>C mutation can cause splicing defects, leading to reduced full-length mRNA in patient fibroblasts and skipping of exons 39–40. Compared to the non-mutant control group, reduced levels of full-length ABCA4 protein can be observed ^[4].

2. Sequencing of ABCA4 cDNA

Using primers specifically paired with human ABCA4 mRNA, reverse transcription was performed to amplify cDNA, followed by gel electrophoresis to determine the expression of the human ABCA4 gene. The result shows that three types of human ABCA4 cDNA bands (Normal mRNA, Exon 39 skipped mRNA, and Exons 39-40 skipped mRNA) were present in the eye tissue of huABCA4-c.5461-10T>C mice, with band types and sizes matching the expected patterns. Sequencing analysis of ABCA4 cDNA reveals that exons 38 to 41 of the mouse Abca4 gene in huABCA4-c.5461-10T>C mice were successfully replaced by exons 38 to 41 of the human ABCA4 gene carrying the c.5461-10T>C mutation, resulting in the expected three distinct transcripts.

Figure 3. Sequencing results of the region from exon 38 to exon 41 of the human ABCA4 gene in huABCA4-c.5461-10T>C mice.

3. Protein Expression

Western Blot results showed bands in both huABCA4 and WT mouse eyes, indicating human ABCA4 protein expression in huABCA4 mice, while the WT band was attributed to high human-mouse homology. In eye tissues of huABCA4-c.5461-10T>C mice, human ABCA4 protein expression was significantly lower than in huABCA4 mice.

Figure 4. Detection of human ABCA4 protein expression in eye tissues of female huABCA4-c.5461-10T>C mice, huABCA4 mice, and wild-type (WT) mice*.
*The WB profile showed double bands near 270KD. Given that ABCA4 is a highly glycosylated protein with multiple N-linked glycosylation sites, the upper band is speculated to result from glycosylation modification.

4. Fundus morphology, OCT images, and retinal thickness analysis

(A) Representative fundus and OCT images of huABCA4 and huABCA4-c.5461-10T>C mice at 4 weeks, 6 months and 11 months of age, showing no obvious abnormalities in either group at all time points.

(B) Quantitative analysis of retinal thickness in huABCA4 and huABCA4-c.5461-10T>C mice at the indicated ages, revealing no significant differences between groups.

Figure 5. Fundus morphology, OCT images, and retinal thickness analysis of huABCA4 and huABCA4-c.5461-10T>C mice at different ages.

5. Electroretinogram (ERG)

(A) At 4 weeks of age, the amplitudes of the a-wave and b-wave in both scotopic and photopic electroretinograms (ERGs) of huABCA4-c.5461-10T>C mice were almost identical to those of huABCA4 mice.

(B) By 6 months of age, the amplitudes of the a-wave and b-wave in scotopic ERG recordings of huABCA4-c.5461-10T>C mice had begun to decrease gradually.

(C) By 11 months of age, the a-wave and b-wave amplitudes in the scotopic ERG recordings of huABCA4-c.5461-10T>C mice were significantly lower than in huABCA4 mice.

Figure 6. Electroretinogram (ERG) detection results of huABCA4 and huABCA4-c.5461-10T>C mice at 4 weeks, 6 months and 11 months of age, respectively.

6. Pathological observation (immunofluorescence staining)

Retinal sections from WT, huABCA4, and huABCA4-c.5461-10T>C mice were analyzed by immunofluorescence staining at 11 weeks of age. ABCA4 expression was clearly detected in the photoreceptor cells of both WT and huABCA4 mice, with no significant difference between these two groups. In contrast, no ABCA4 expression was observed in the retinas of huABCA4-c.5461-10T>C mice, confirming the successful mutation and loss of ABCA4 protein in this group.

Figure 7. ABCA4 mutation leads to the loss of ABCA4 expression in the retinas of huABCA4-c.5461-10T>C mice.

huABCA4-c.5461-10T>C Mouse

基本情報

検証 Data

関連リソース

遺伝子名

遺伝子別名

NCBI ID

染色体

MGI ID

さらに

データシート

系統詳細

参考文献

系統作製戦略

適用分野

1. Expression of human ABCA4 and mouse Abca4 genes

2. Sequencing of ABCA4 cDNA

3. Protein Expression

4. Fundus morphology, OCT images, and retinal thickness analysis

5. Electroretinogram (ERG)

6. Pathological observation (immunofluorescence staining)

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