B6-hMECP2 Mice

Catalog Number: C001568

Strain Name: C57BL/6NCya-Mecp2tm1(hMECP2)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

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

Strain Description

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that occurs predominantly in female infants and young children. The incidence is approximately 1 in 10,000–15,000 females. Clinical features include intellectual disability, loss of language function, stereotyped hand movements, and gait abnormalities. Affected children typically have a period of normal development followed by stagnation of head circumference growth at 6–18 months of age, and regression of acquired skills. Overt cognitive and motor impairments develop 1–2 years later. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for >90% of RTT cases. MECP2 is a nuclear protein that binds to methylated DNA to regulate gene transcription. MECP2 duplications cause MECP2 duplication syndrome (MDS), while functional deficiency of MECP2 impairs the production of this nuclear protein, leading to central nervous system functional maturation disorders that affect learning and memory functions, resulting in RTT.

Treatment for RTT focuses mainly on gene supplementation therapy based on adeno-associated virus (AAV) vectors. This involves delivering human MECP2 genes via AAV vectors to compensate for the deficiency of MECP2 genes in patients. However, the large size of the MECP2 gene exceeds the delivery capacity of most vectors, and over-expression of the MECP2 gene can also lead to serious neurological diseases. These limitations have hindered the development of this therapy. Therefore, DNA/RNA editing to repair MECP2 gene mutations and restore normal expression of MECP2 protein has received widespread attention. Currently, multiple research groups have used CRISPR-based gene editing technology to repair mutations in the MECP2 gene in induced pluripotent stem cells (iPSCs) or ex vivo patient cells [1,2]. Animal studies are an essential part of preclinical research. RTT therapies based on small nucleic acids, CRISPR gene editing technology, base editors, and RNA editing technology target the human MECP2 gene. Humanized mouse models can help advance gene therapy drug pipelines into clinical stages.

This strain is a humanized MECP2 gene mouse model that can be used for RTT research. Homozygous B6-hMECP2 mice are viable and fertile. Additionally, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also generate hot mutation models based on this strain (B6-hMECP2*T158M, Catalog Number: C001569) and provide customized services for specific mutations to meet experimental needs in pharmacology and other RTT-related fields.

Strain Strategy

Figure 1. Gene editing strategy of B6-hMECP2 mice. The mouse Mecp2 endogenous domain (aa.10~484) was replaced with the human MECP2 domain (aa.10~486). The mouse Mecp2 domain (aa.1-9) and the human MECP2 domain (aa.1-9) encode the same sequence.

Application

  • Research on Rett syndrome (RTT)

 

Validation Data

1. Human MECP2 and mouse Mecp2 gene expression


Figure 2. Gene expression in the brain of 8-week-old wild-type (WT), B6-hMECP2 (hemizygous males hMeCP2KI/y, homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y, heterozygous females hMeCP2T158M/+). RT-qPCR results indicate that the human MECP2 gene is significantly expressed in the brains of B6-hMECP2 and B6-hMECP2*T158M mice, while it is not expressed in WT mice. The mouse Mecp2 gene is significantly expressed in the brains of WT mice, but not in homozygous B6-hMECP2 or B6-hMECP2*T158M mice. (ND: Not detected; Bars represent mean ± SEM; n≥3)

2. Survival curves


Figure 3. Survival curves of wild-type (WT), B6-hMECP2 (hemizygous males hMeCP2KI/y, homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y, heterozygous females hMeCP2T158M/+).

3. Growth and Phenotype Observations

1) Hemizygous Males


Figure 4. Body weight, length, and phenotypic scores of wild-type (WT), B6-hMECP2 (hemizygous males hMeCP2KI/y), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y). The results show that the body weight and length of the hemizygous males hMeCP2T158M/y are significantly lower than those of the other groups. From 4 weeks of age, these mice begin to exhibit mild RTT phenotypes, which become noticeable at 6 weeks of age. As the mice age, the phenotype gradually worsens. (Bars represent mean ± SEM)


Figure 5. Images of 8-9-week-old wild-type (WT), B6-hMECP2 (hemizygous males hMeCP2KI/y), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y). At 8-9 weeks of age, approximately 70% of hMeCP2T158M/y mice exhibit hindlimb clasping, with a few severe cases showing bilateral hindlimb clasping. Occasionally, some WT and hMeCP2KI/y mice also exhibit hindlimb clasping.


Figure 6. Images of 13-week-old wild-type (WT), B6-hMECP2 (hemizygous males hMeCP2KI/y), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y). The hMeCP2T158M/y mice show obvious head abnormalities, with a smaller head circumference. Their heads and mouths are noticeably shorter compared to other groups, and they have coarse fur, poor overall condition, and dull eyes. In contrast, the hMeCP2KI/y mice have a normal head circumference, pointed mouths, smooth and shiny fur, and bright, lively eyes.

2) Homozygous Females


Figure 7. Body weight, body length, and phenotypic scores of wild-type (WT), B6-hMECP2 (homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (homozygous females hMeCP2T158M/T158M). The body weight and body length of hMeCP2T158M/T158M mice are significantly lower than those of the other groups. From 4 weeks of age, these mice begin to exhibit mild RTT phenotypes, which become noticeable at 6 weeks of age. As the mice age, the phenotype gradually worsens. (Bars represent mean ± SEM)


Figure 8. Images of 8-9-week-old wild-type (WT), B6-hMECP2 (homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (heterozygous females hMeCP2T158M/+). At 8-9 weeks of age, approximately 30% of hMeCP2T158M/+ mice exhibit unilateral hindlimb clasping. Occasionally, some WT and hMeCP2KI/KI mice also exhibit hindlimb clasping.

Figure 9. Images of 13-week-old wild-type (WT), B6-hMECP2 (homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (heterozygous females hMeCP2T158M/+). No obvious head abnormalities have been observed in hMeCP2T158M/+ mice thus far. The hMeCP2KI/KI mice have a normal head circumference, pointed mouths, smooth and shiny fur, and bright, lively eyes.

4. Grip strength test & Rotarod test


Figure 10. The grip strength and rotarod analysis of hMeCP2 mice at 5 to 6-week-old (A) and 11 to 12-week-old (B).

①Grip Strength Test: All hMeCP2 mice exhibited a decline in grip strength compared to WT

②Rotarod Test: Rotarod performance revealed that both male hMeCP2 T158M hemizygous mice and female hMeCP2 T158M homozygous mice exhibited reduced latency compared to WT and hMeCP2 control mice.

This reduction suggests impaired locomotor activity and coordination in the mutant mice.

5. Gait Analysis


Figure 11.The gait analysis of hMeCP2 mice at 5 to 6-week-olds (A) and 11 to 12-week-olds (B).

Upon integration of the hindlimb stride length and width - we see the genotype-based severity of the pathology corresponds to an increased hindlimb stride width with decreased hindlimb stride length (shorter but wider steps.) By comparison, we could see that female and male hMeCP2T158M homo/hemi show the most severe phenotype, while male hMeCP2T158M/y and mixed-sex hMeCP2WT/WT are similar to each other and close to WT control. The female hMeCP2T158M/+ mice are between these two clusters. Over time, this pathology - shorter but wider steps - has become more evident.

6. Conclusion

These data reveal the weakening of muscle strength, the decrease of coordination, the decline of exercise efficiency, as well as the deterioration of gait health status in hMeCP2 T158M mice.

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the MECP2 gene

https://rddc.tsinghua-gd.org/en/gene/4204


2. MECP2 clinical variants

 

3. Disease introduction

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder that occurs predominantly in female infants and young children, with an incidence of approximately 1/10,000 to 1/15,000 females. Clinical features include intellectual disability, loss of language function, stereotyped hand movements, and gait abnormalities. Affected children typically have normal development in the early stages, followed by stagnation of head circumference growth at 6–18 months of age and regression of acquired skills. Overt cognitive and motor impairments develop 1–2 years later. Mutations in the methyl-CpG binding protein 2 (MECP2) gene account for >90% of RTT cases. Normally, females have one functional MECP2 copy on each X chromosome; however, in most cases of RTT, patients have only one mutated MECP2 copy among their two copies. This is because X chromosome inactivation in neurons silences the other normal MECP2 copy, resulting in insufficient MECP2 protein expression and RTT [1].

4. MECP2 gene and mutations

The MECP2 gene is located on the X chromosome and encodes a nuclear protein that binds to methylated DNA to regulate gene transcription and expression. Repetitive mutations in MECP2 can cause MECP2 duplication syndrome (MDS), while functional deficiency of MECP2 can impair production of this nuclear protein, leading to central nervous system functional maturity disorders that affect learning and memory functions and cause Rett syndrome (RTT). Over 100 MECP2 gene mutations have been identified to date, with more than 80% being cytosine-to-thymine (C>T) transitions. Hotspot mutations, which account for 70% of all mutations, include R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C. T158M is the most common mutation type.

5. Function of non-coding DNA sequences

Research indicates that pathogenic mutations exist in the introns of the MECP2 gene [3].

6. MECP2-targeted gene therapy

RTT treatment mainly focuses on MECP2 gene supplementation therapy based on AAV vectors. This involves delivering human MECP2 genes through AAV vectors to compensate for the deficiency of MECP2 genes in patients. However, the large size of the MECP2 gene exceeds the delivery capacity of most vectors, and overexpression of the MECP2 gene can also lead to serious neurological diseases, limiting the development of this therapy. Therefore, gene/RNA editing to repair MECP2 gene mutations and restore normal expression of MECP2 protein has received widespread attention. Currently, multiple research groups have used CRISPR-based gene editing technology to repair mutations in the MECP2 gene in induced pluripotent stem cells (iPSCs) or ex vivo patient cells [1,2].

Additionally, some studies have used transgenic humanized mice for in vivo pharmacological evaluations. For example, researchers have shown that human-specific MECP2-ASO drugs can effectively downregulate the overexpression of MECP2 in the brains of transgenic humanized MDS mice (Mecp2-/Y; MECP2-TG1; MECP2-GFP). The drug can alleviate various behavioral defects caused by excessive expression of MECP2 and restore normal expression in a dose-dependent manner [4]. However, transgenic humanized mice used in such in vivo experiments have defects, such as complex construction, insufficient copy number, random insertion, and insufficient humanization region. More efficient in vivo gene editing models are yet to be developed.

7. Summary

Rett syndrome (RTT) is a severe neurological disorder that arises from mutations in the MECP2 gene. Humanized MECP2 mice serve as an invaluable resource for conducting preclinical research on RTT and for the development of gene therapy drugs. Cyagen provides whole-genome humanized MECP2 mouse models, which carry the entire human MECP2 gene and can produce humanized MECP2 mice with specific point mutations to facilitate the study of RTT pathogenesis and the development of therapies targeting MECP2.

 

 

References
[1]Qian J, Guan X, Xie B, et al. Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons[J]. Science Translational Medicine, 2023, 15(679): eadd4666.
[2]Thi T H, Tran N T, Mai T, et al. Efficient and precise CRISPR/Cas9-mediated MECP2 modifications in human induced pluripotent stem cells[J].Frontiers in Genetics, 2019, 10.
[3]Amir, R E. Mutations in exon 1 of MECP2 are a rare cause of Rett syndrome[J]. Journal of Medical Genetics, 2005, 42(2):e15.
[4]Shao Y, Sztainberg Y, Wang Q, Bajikar SS, Trostle AJ, Wan YW, Jafar-Nejad P, Rigo F, Liu Z, Tang J, Zoghbi HY. Antisense oligonucleotide therapy in a humanized mouse model of MECP2 duplication syndrome. Sci Transl Med. 2021 Mar 3;13(583):eaaz7785.