Mecp2R168X is one of the most common MeCP2 mutations associated with Rett syndrome (RTT). Mecp2R168X mice express a truncated R168X protein with a partial NLS and lacking the transcriptional repression domain (TRD) for interaction with corepressors. This RTT model is useful for testing highly robust behavioral paradigms in preclinical drug trials.
Laura R Schaevitz, Tufts University
The early truncating MeCP2R168X nonsense point mutation in the methyl-CpG binding domain of MeCP2 is one of the most common MeCP2 mutations associated with Rett syndrome (RTT). The largely truncated R168X protein retains the capacity to bind methylated DNA and carries a partial nuclear localization signal, but lacks the transcriptional repression domain for interaction with co-repressors. A second termination (BspH1 restriction site) downstream of the R168X mutation is reported not to affect the mutant product. MeCP2R168X mice are a model of RTT; exhibiting respiratory, neuromuscular and behavioral abnormalities similar, but not identical, to that of Mecp2 null mice. The phenotype is described in detail below. These mice also underscore the importance of including Mecp2 mutant females in preclinical studies.
MeCP2R168X mice on a mixed genetic background of ~87-93% C57BL/6J and ~13-6% 129S6/SvEvTac (B6J;129S6.MeCP2R168X) are Stock No. 024990. In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype of these B6J;129S6.MeCP2R168X mice could vary from that originally described on other genetic backgrounds. We will modify the strain description if necessary as published results become available.
Lawson-Yeun et al. 2007 Brain Res 1180:1 reports the phenotype of Mecp2R168X animals backcrossed at least 10 generation onto the 129S6/SvEvTac genetic background (129S6.Mecp2R168X) as: hemizygous males (MeCP2R168X/Y) have a shortened lifespan (average 86 days) with forelimb stereotypies, hindlimb atrophy, hypoactivity and breathing irregularities. By 7 weeks of age, significant hindlimb clasping is evident. Female heterozygotes (MeCP2R168X/+) manifest significant defects by approximately 6 months (hindlimb clasping, breathing irregularities) and can survive past 1 year of age.
Schaevitz et al. 2013 Genes Brain Behav 12:732 reports the phenotype of 129S6.Mecp2R168X animals backcrossed 1-2 generations onto C57BL/6J as: MeCP2R168X mutants mirror many clinical features of human RTT. MeCP2R168X/Y males exhibit growth, motor, respiratory and cognitive abnormalities, and reduced anxiety. The phenotype is less severe and with later onset in MeCP2R168X/+ females with the exception of seizures; ~4% of MeCP2R168X/+ females exhibit tonic-clonic seizures that typically result in death. The phenotype in MeCP2R168X/Y males is similar to that reported for Mecp2 null males (such as Mecp2tm1.1Bird; Stock No. 003890). Compared to females heterozygous for the Mecp2 null mutation, MeCP2R168X/+ females exhibit delayed motor defect onset, normal anxiety-like behavior and increased seizure susceptibility.
Bissonnette et al. 2014 Neuroscience 267:166 reports the phenotype of Mecp2R168X animals on a mixed C57BL/6J;129S6/SvEvTac genetic background as: MeCP2R168X/+ females display augmented hypoxic ventilatory responses and depressed hypercapnic responses: the incidence of apnea is much greater in MeCP2R168X/+ females (189 per hour) than MeCP2T158A/+ females (41 per hour).
For B6J;129S6.MeCP2R168X mice, the donating investigator reports that mating hemizygous males with wildtype females does not produce offspring; it is not known if the hemizygous males are sterile or if they simply do not breed as a result of their Rett syndrome-like phenotype. The donating investigator also reports that backcrossing MeCP2R168X mice onto the C57BL/6 genetic background leads to increased phenotype severity.
The phenotype differences observed between male and female mice is because Mecp2 is located on the X chromosome. Due to X-chromosome inactivation, heterozygous females have mosaic expression of wildtype MeCP2.
To create the targeting vector, Dr. Joseph T. Coyle (Harvard Medical School, McLean Hospital) isolated a mouse methyl-CpG binding protein 2 (Mecp2) DNA sequence and used site-directed mutagenesis to introduce an AGA-->TGA (arginine-->stop) substitution into amino acid 168 in exon 4. The nonsense point mutation creates an R168X amino acid substitution in the methyl-CpG binding domain of MeCP2, one of the most common MeCP2 mutations associated with Rett syndrome. The targeting vector also inserted a loxP-flanked neomycin resistance cassette (neo), as well as inserted a second termination (BspH1 restriction site) downstream of the R168X mutation.
This construct was targeted to the Mecp2 gene on the X chromosome via electroporation into 129/SvJ-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts, and chimeric mice were bred to 129S6/SvEvTac mice. Offspring with germline transmission of R168X were bred with Cre recombinase expressing mice (undisclosed genetic background) to remove the floxed neo cassette. The resulting mice harboring the Mecp2R168X allele were maintained on a 129S6/SvEvTac genetic background. Drs. Joanne E. Berger-Sweeney and Laura R. Schaevitz (Tufts University) obtained the mice. There, the colony was bred three generations onto C57BL/6J. Because the phenotype was more severe with successive backcrossing onto C57BL/6J, the Mecp2R168X colony was thereafter maintained by breeding heterozygous females with wildtype males from the colony. Males from the B6J;129S6.Mecp2R168X colony (~87.5% B6J and ~12.5% 129S6) were sent to The Jackson Laboratory in 2014. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664). Thereafter, the live B6J;129S6.Mecp2R168X colony was maintained by breeding heterozygous females with wildtype males from the colony.
|Allele Name||targeted mutation 1.1, Joseph T Coyle|
|Allele Type||Targeted (Humanized sequence)|
|Gene Symbol and Name||Mecp2, methyl CpG binding protein 2|
|Strain of Origin||129/SvJ|
|Molecular Note||The targeting vector was created by using site-directed mutagenesis to introduce an AGA-->TGA (arginine-->stop) substitution into amino acid 168 in exon 4. The nonsense point mutation creates an R168X amino acid substitution in the methyl-CpG binding domain. This mutation corresponds to one of the most common MeCP2 mutations associated with human Rett syndrome. The targeting vector also inserted a loxP-flanked neomycin resistance cassette (neo). Cre-mediated recombination removed the floxed neo cassette. The substitution was confirmed by sequencing.|
The MeCP2R168X mutant allele is located on the X chromosome. Hemizygous males exhibit features of Rett syndrome and have a shortened lifespan. Heterozygous females may live more than one year and have a less severe phenotype with later onset. When maintaining a live colony, heterozygous females may be bred with wildtype males from the colony.
The donating investigator reports that mating hemizygous males with wildtype females does not produce offspring; it is not known if the hemizygous males are sterile or if they simply do not breed as a result of their Rett syndrome-like phenotype. The donating investigator also reports that backcrossing MeCP2R168X mice onto the C57BL/6 genetic background leads to increased phenotype severity. The expected coat colors are agouti and black.
When using the B6J;129S6.MeCP2R168X mouse strain in a publication, please cite the originating article(s) and include JAX stock #024990 in your Materials and Methods section.