Name: huUSH2A(E10-15)-c.2299delG Mouse
Brand: サイヤジェン（Cyagen）
SKU: C001850

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huUSH2A(E10-15)-c.2299delG Mouse

製品名

huUSH2A(E10-15)-c.2299delG Mouse

製品ID

C001850

系統名

C57BL/6JCya-Ush2a^{tm2(hUSH2A*c.2299delG)}/Cya

背景情報

C57BL/6JCya

状況

このマウス系統を論文で使用する場合は、「huUSH2A(E10-15)-c.2299delG Mouse（カタログ番号C001850）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

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遺伝子型

性別

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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

The USH2A gene encodes Usherin, a protein featuring laminin EGF-like, pentraxin, and fibronectin type III domains, predominantly expressed in the basement membrane of the inner ear and retina. Usherin plays a critical role in developing hair cells in the inner ear, auditory signal transduction, and the maintenance of adhesion via interactions with fibronectin in the retinal basement membrane. Mutations in the USH2A gene disrupt the normal development and function of hair cells, impair fibronectin assembly, and compromise the adhesive properties of the retinal basement membrane, leading to hearing loss and RP symptoms. The USH2A gene is the primary causative gene for Usher syndrome Type II (USH2), with 75%–90% of USH2 cases linked to mutations in this gene ^[1]. Exon 13 of the USH2A gene harbors a hotspot for pathogenic mutations associated with USH, including two common mutations, c.2299delG and c.2276G>T, which are the subject of several therapeutic investigations. The c.2299delG mutation in exon 13 causes frameshift and premature termination codons. This mutation produces a protein that is 85% truncated compared to the normal transcript size, leading to the loss of Usherin protein function ^[2-5]. Currently, there are no effective therapies for Usher syndrome. Ongoing research focuses on elucidating the genetic mechanisms underlying the disorder and developing gene-based therapeutic strategies.

huUSH2A(E10-15)-c.2299delG mice are obtained by introducing the c.2299delG mutation into the exon 13 of the human USH2A gene in huUSH2A(E10-15) mice (Catalog Number: C001554) using gene editing technology. This model can be used to study the mechanisms and therapeutic approaches for diseases such as Usher syndrome Type II.

参考文献

McGee TL, Seyedahmadi BJ, Sweeney MO, Dryja TP, Berson EL. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa. J Med Genet. 2010 Jul;47(7):499-506.

Pendse ND, Lamas V, Pawlyk BS, Maeder ML, Chen ZY, Pierce EA, Liu Q. In Vivo Assessment of Potential Therapeutic Approaches for USH2A-Associated Diseases. Adv Exp Med Biol. 2019;1185:91-96. doi: 10.1007/978-3-030-27378-1_15. PMID: 31884594.

Yan D, Ouyang X, Patterson DM, Du LL, Jacobson SG, Liu XZ. Mutation analysis in the long isoform of USH2A in American patients with Usher Syndrome type II. J Hum Genet. 2009 Dec;54(12):732-8. doi: 10.1038/jhg.2009.107. Epub 2009 Oct 30. PMID: 19881469; PMCID: PMC4511341.

Dreyer B, Tranebjaerg L, Brox V, Rosenberg T, Möller C, Beneyto M, Weston MD, Kimberling WJ, Cremers CW, Liu XZ, Nilssen O. A common ancestral origin of the frequent and widespread 2299delG USH2A mutation. Am J Hum Genet. 2001 Jul;69(1):228-34. doi: 10.1086/321269. Epub 2001 Jun 8. Erratum in: Am J Hum Genet 2001 Oct;69(4):922. PMID: 11402400; PMCID: PMC1226039.

Dulla K, Slijkerman R, van Diepen HC, Albert S, Dona M, Beumer W, Turunen JJ, Chan HL, Schulkens IA, Vorthoren L, den Besten C, Buil L, Schmidt I, Miao J, Venselaar H, Zang J, Neuhauss SCF, Peters T, Broekman S, Pennings R, Kremer H, Platenburg G, Adamson P, de Vrieze E, van Wijk E. Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations. Mol Ther. 2021 Aug 4;29(8):2441-2455. doi: 10.1016/j.ymthe.2021.04.024. Epub 2021 Apr 23.

系統作製戦略

The start codon (ATG) of the mouse Ush2a gene is located in exon 1, whereas the start codon (ATG) of the human USH2A gene resides in exon 2. Consequently, exons 10–15 of the human USH2A gene correspond to exons 9–14 of the mouse Ush2a gene. Exons 9 to 14 of the mouse Ush2a gene and their flanking sequences were replaced by exons 10 to 15 of the human USH2A gene and their respective flanking sequences. The c.2299delG point mutation was introduced into exon 13 of human USH2A.

Figure 1. Gene editing strategy for the huUSH2A(E10-15)-c.2299delG mouse model.

適用分野

Investigation of the pathogenic mechanisms underlying Usher syndrome (USH) and preclinical evaluation of therapeutic drugs;

Development, screening, and preclinical evaluation of drugs targeting USH2A.

検証 Data

1. Sequencing Analysis

Total RNA was extracted from eye tissues of 6‑week‑old heterozygous huUSH2A(E10-15)‑c.2299delG mice (n=2) and reverse‑transcribed into cDNA. PCR primers were designed in human exon 12 and mouse exon 16 to specifically amplify the human USH2A transcript. Electrophoresis showed two distinct bands, indicating the coexistence of two alternatively spliced RNA products.

cDNA sequencing of the PCR products clearly identified two transcript isoforms, corresponding to the E13‑containing and E13‑skipped splicing patterns, respectively. These results are highly consistent with the model design and splicing predictions.

Figure 2. Schematic diagram of gene expression.

Figure 3. cDNA sequencing results of eye tissues from huUSH2A‑c.2299delG mice (6 weeks old, heterozygous, n=2).

2. Gene Expression

To detect the expression levels of human USH2A and mouse Ush2a transcripts in Eye tissues of huUSH2A(E10-15), huUSH2A(E10-15)-c.2299delG, and wild-type (WT) mice, three pairs of specific primers were used for RT-qPCR analysis. mGapdh was selected as the internal reference gene to calculate the relative gene expression levels. All data are presented as mean ± standard deviation (mean±SD). Statistical analysis was performed using one-way ANOVA. Significance was defined as follows: ns, no significant difference; ***, p < 0.001.

RT-qPCR analysis confirmed the presence of human USH2A transcripts, but not murine Ush2a transcripts, in the eye tissues of both huUSH2A(E10-15) and huUSH2A(E10-15)-c.2299delG mice (Figs. A and B). Conversely, murine Ush2a transcripts were detected in WT mice, while human USH2A transcripts were absent (Fig. C).

Figure 4. Detection of the transcriptional levels of human USH2A (Figure A and Figure B) and murine Ush2a (Figure C) in the eye tissues of huUSH2A(E10-15)-c.2299delG mice, huUSH2A(E10-15) mice, and wild-type (WT) mice by RT-qPCR (5-week-old, male, n=4).
Figure A: The primers targeted the E14-E15 exons of the human USH2A sequence. The results showed that the mRNA expression level of human USH2A in the eye tissues of huUSH2A(E10-15)-c.2299delG mice was similar to that in huUSH2A(E10-15) mice, and there was no significant difference (ns) between the groups.
Figure B: The primers targeted the E13 exon of the human USH2A sequence. The results showed that compared with huUSH2A(E10-15) mice, the mRNA expression level of human USH2A in the eye tissues of huUSH2A(E10-15)-c.2299delG mice decreased significantly (***p < 0.001). Combined with the analysis of RNA sequencing results, this phenomenon occurred because after the mutation in huUSH2A(E10-15)-c.2299delG mice, some human USH2A transcripts underwent skipping splicing and skipped the E13 exon.
Figure C: The primers targeted the E12 exon of the murine Ush2a sequence for detection. The results showed that the murine Ush2a transcripts could be clearly detected in the eye tissues of wild-type (WT) mice, while the human USH2A transcripts were not detected, showing a significant difference from the previous two groups of mice.

huUSH2A(E10-15)-c.2299delG 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

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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. Sequencing Analysis

2. Gene Expression

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