STOCK Mecp2^tm4.1Joez/J

Stock number: 029643
Product name: Mecp2-R106W-Tavi
Research areas: Developmental Biology Research, Mouse/Human Gene Homologs, Neurobiology Research, Reproductive Biology Research

Description

Mecp2-R106W-Tavi knock-in mice carry a missense mutation (R106W) and biotinylation consensus motif in the X-linked Mecp2 gene, and are useful when studying the neurodevelopmental disorder Rett syndrome.

Detailed description

These Mecp2-R106W-Tavi mice contain a missense mutation in exon 3 of the endogenous X-linked methyl-CpG binding protein 2 (Mecp2) gene. This mutation introduces the amino acid mutation R106W in the methyl-CpG binding domain (MBD) of MeCP2, analogous to the common missense mutation found in humans with Rett Syndrome (RTT), an X-linked neurological disorder that can be caused by mutations in the MECP2 gene. A 23-amino acid affinity tag was also inserted immediately upstream of the Mecp2 stop codon. This tag comprises a TEV protease cleavage site and a 15-amino acid biotinylation consensus motif (termed Tavi, TEV and Avidin-binding) that can be post-translationally labeled with biotin in the presence of the E. coli biotin ligase, BirA. Hemizygous males with the R106W mutation exhibit RTT-like phenotypes after 5-6 weeks of age. They are infertile and have a shortened lifespan with median survival of ~10 weeks. Heterozygous females are viable and fertile. Due to X-chromosome inactivation, heterozygous females have mosaic expression of wild-type MeCP2.

When bred to cBirA mice (Stock No. 030420), with Cre-dependent BirA expression, and subsequently bred to a cre-expressing line, Mecp2-R106W-Tavi is reliably biotinylated in Cre-defined cells/tissues.

Of note, Mecp2-Tavi (Stock No. 030422) and Mecp2-T158M-Tavi (Stock No. 029642) knock-in companion strains are also available.

Development

A targeting construct was designed to insert a loxP-flanked neomycin (neo) resistance cassette upstream of exon 4 of the X-linked methyl-CpG binding protein 2 (Mecp2) gene. A missense mutation R106W found in humans carrying the neurological disorder Rett Syndrome (RTT) was introduced to exon 3. A 23 amino acid Tavi tag, allowing MeCP2 protein to be biotinylated in the presence of E. coli biotin ligase, BirA, was introduced immediately upstream of Mecp2 stop codon. This targeting construct was electroporated into 129Sv-derived embryonic stem (ES) cells and correctly targeted ES cells were injected into C57BL/6 blastocysts. Chimeric males were bred to C57BL/6 females and offspring were bred to B6.FVB-Tg(EIIa-cre)C5379Lmgd/J transgenic mice (Stock No. 003724) to remove the neo cassette. The resulting offspring were backcrossed to C57BL/6J mice (Stock No. 000664) for at least 10 generations to establish a colony of Mecp2-R106W-Tavi knock-in mice. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Due to fertility issues on the C57BL/6J background, an aliquot of frozen sperm was used to fertilize 129S1/SvImJ oocytes (Stock No. 002448) to establish our live colony.

Allele

Symbol: Mecp2^tm4.1Joez
Name: targeted mutation 4.1, Zhaolan Zhou
MGI accession ID: MGI:5790796
Generation method: Targeted
Attributes: Humanized sequence; Epitope tag
Synonyms: Mecp2^R106W-Tavi; R106W
Marker symbol: Mecp2
Marker name: methyl CpG binding protein 2
Marker synonyms: 1500041B07Rik; AUTSX3; D630021H01Rik; Mbd5; MECP2_e1; MRX16; MRX79; MRXS13; MRXSL; PPMX; RS; RTS; RTT; WBP10
Chromosome: X
Strain of origin: 129
Molecular note: A targeting construct was designed to insert a loxP-flanked neomycin (neo) resistance cassette upstream of exon 4 of the gene. A missense mutation R106W found in humans carrying the neurological disorder Rett Syndrome (RTT) was introduced to exon 3. A 23 amino acid Tavi tag, allowing MeCP2 protein to be biotinylated in the presence of E. coli biotin ligase, BirA, was introduced immediately upstream of Mecp2 stop codon. Cre-mediated recombination removed the upstream floxed neo cassette.