B6-hMECP2*T158M Mice
Catalog Number: C001569
Strain Name: C57BL/6NCya-Mecp2tm2(hMECP2*T158M)/Cya
Genetic Background: C57BL/6NCya
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 primarily affecting female infants and young children, with an incidence of approximately 1 in 10,000 to 15,000 females. Characteristic clinical features include intellectual disability, loss of language skills, stereotypic hand movements, and gait disturbances. Affected individuals typically experience a period of normal development, followed by deceleration in head circumference growth between 6 to 18 months of age, and subsequent regression of acquired motor and cognitive abilities. Overt impairments in cognition and motor function generally emerge within 1 to 2 years. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene are responsible for over 90% of RTT cases. MECP2 is a nuclear protein that binds methylated DNA to modulate gene transcription. MECP2 gene duplications lead to MECP2 duplication syndrome (MDS), while MECP2 deficiency disrupts central nervous system maturation, adversely affecting learning and memory, culminating in the clinical manifestations of RTT.
Current therapeutic strategies for RTT primarily revolve around gene supplementation using adeno-associated virus (AAV) vectors to deliver functional human MECP2 genes to compensate for the endogenous deficiency. However, the substantial size of the MECP2 gene surpasses the packaging capacity of most viral vectors, and overexpression of MECP2 poses a risk of severe neurological complications. These challenges have significantly impeded the progress of gene supplementation therapies. Consequently, the focus has shifted towards DNA/RNA editing approaches aimed at correcting MECP2 mutations and restoring physiological levels of MECP2 protein expression. Notably, several research groups have successfully employed CRISPR-based gene editing technologies to rectify MECP2 mutations in induced pluripotent stem cells (iPSCs) or patient-derived cells ex vivo [1,2]. Given the pivotal role of animal models in preclinical research, the development of humanized mouse models expressing the human MECP2 gene is crucial. These models facilitate the transition of gene therapy candidates—encompassing small nucleic acids, CRISPR-based editors, base editors, and RNA editing technologies—into clinical stages.
This strain is a humanized MECP2 gene mouse model, generated by replacing the endogenous mouse Mecp2 gene with the human MECP2 gene harboring the T158M mutation through embryonic stem cell targeting techniques. This mutation represents the most common human RTT-associated missense mutation in MECP2. Studies have shown that mice carrying this mutation recapitulate many clinical features of RTT [5].
Strain Strategy
Figure 1. Gene editing strategy of B6-hMECP2*T158M 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. The point mutation T158M (ACG to ATG) was introduced into the human MECP2 exon 4.
Application
B6-hMECP2*T158M mice can serve as a valuable model for studying the mechanisms of RTT and could potentially be used to develop or validate targeted therapies.
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/+, homozygous females hMeCP2T158M/T158M). The hMeCP2T158M/y mice exhibit severe mortality, with only one mouse surviving up to 24 weeks. The hMeCP2T158M/T158M mice show relatively better survival, with approximately 50% survival rate up to 24 weeks. There may be slight variations in survival rates among different batches of mice.
3. Immunofluorescence (IF) staining
Figure 4. Immunofluorescence (IF) staining of 16-week-old hemizygous male B6-hMECP2*T158M mice and wild-type (WT, C57BL/6JCya) mice.
*The injected phebAAV-hsyn-EGFP is a virus carrying a fluorescent tag and has no therapeutic effect.
*This data was provided by the Cyagen customer.
4. Pathological analysis
1) Brain weight
Figure 5. Brain weight of wild-type (WT) and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y, heterozygous females hMeCP2T158M/+). (**p<0.01; ***p<0.001; Bars represent mean ± SEM; n=4).
2) HE staining & Nissl staining
Figure 6. HE staining & Nissl staining of 23-week-old wild-type (WT) and hemizygous male B6-hMECP2*T158M mice (hMeCP2T158M/y).
① HE Staining Results: Hemizygous male B6-hMECP2*T158M mice do not show significant tissue structure abnormalities, and neuronal necrosis or degenerative changes are not evident.
② Nissl Staining Results: Hemizygous male B6-hMECP2*T158M mice do not display significant neurological lesions, except for an increased density of pyramidal cell layers in the hippocampal region.
③ At the same magnification (scale bar 500㎛), the overall hippocampal area in hMeCP2T158M/y mice is significantly smaller than that in WT mice. The hippocampus in hMeCP2T158M/y mice is round, whereas, in WT mice, it is normally oval-shaped.
3) Silver Staining
Figure 7. Silver staining of 23-week-old wild-type (WT) and hemizygous male B6-hMECP2*T158M mice (hMeCP2T158M/y).
① Silver staining results help compare the neuronal cell bodies and dendritic axons in the cerebral cortex near the olfactory bulb. WT mice show well-developed, pyramidal-shaped neuronal cell bodies and a large number of formed axons (indicated by red arrows). In hMeCP2T158M/y mice, some neuronal cell bodies appear oval, smaller in size, with significantly fewer, thinner, and shorter axons.
② Compared to WT mice, the overall silver staining in hMeCP2T158M/y mice is lighter. High-magnification results show that their neuronal cell bodies and axons are underdeveloped.
5. Growth and Disease Phenotype Observations
1) Hemizygous Males
Figure 8. Body weight, length, and phenotype scores of wild-type (WT) mice, B6-hMECP2 mice (hemizygous males hMeCP2KI/y), and B6-hMECP2*T158M mice (hemizygous males hMeCP2T158M/y). The results show that the body weight and length of 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 disease phenotype gradually worsens. (Bars represent mean ± SEM)
Figure 9. 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 some severe cases showing bilateral hindlimb clasping. Occasionally, some WT and hMeCP2KI/y mice also exhibit hindlimb clasping.
Figure 10. 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 exhibit obvious head abnormalities, with a smaller head circumference. Their heads and mouths are noticeably shorter compared to the 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) Heterozygous Females
Figure 11. Body weight, body length, phenotype scores, and obesity rate of wild-type (WT) and B6-hMECP2*T158M mice (heterozygous females hMeCP2T158M/+). The results show that around 3 weeks of age, the body weight of hMeCP2T158M/+ mice is relatively low (possibly related to birth dates). By around 7 weeks of age, their body weight is comparable to that of the control group mice, but their body length is significantly shorter than that of WT and hMeCP2KI/KI mice. Mild phenotypes start to appear from 8 weeks of age. By around 27 weeks of age, the obesity rate of hMeCP2T158M/+ mice reaches 80%*. (Bars represent mean ± SEM)
*Obesity is defined as a body weight 20% higher than the average weight of WT mice.
Figure 12. 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 hindlimb clasping, all of which are unilateral. Occasionally, some WT and hMeCP2KI/KI mice also exhibit hindlimb clasping.
Figure 13. Images of 13-week-old wild-type (WT), B6-hMECP2 (homozygous females hMeCP2KI/KI), and B6-hMECP2*T158M mice (heterozygous females hMeCP2T158M/+). There is no significant difference in head circumference between hMeCP2T158M/+ and WT mice at 13 weeks. The hMeCP2KI/KI mice have a normal head circumference, pointed mouths, smooth and shiny fur, and bright, lively eyes.
6. Grip strength test & Rotarod test
Figure 14. The grip strength and rotarod analysis of hMeCP2 mice at 5 to 6-week-old (A) and 11 to 12-week-old (B).
① Grip Strength Test: All hMeCP2 mice exhibited a decline in grip strength compared to WT
② Rotarod 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.
7. Gait Analysis
Figure 15.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.
8. 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.
[5]Lamonica JM, Kwon DY, Goffin D, Fenik P, Johnson BS, Cui Y, Guo H, Veasey S, Zhou Z. Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome-like phenotypes. J Clin Invest. 2017 May 1;127(5):1889-1904.
