STOCK Gt(ROSA)26Sor^{tm1.1(birA)Mejr} Mef2c^tm1.1Wtp/Mmjax

Stock number: 025983
Product name: Rosa26^BirA; Mef2c-flbio
Research areas: Research Tools

Description

In these double mutant mice, MEF2C (myocyte enhancer factor 2C) is biotinylated, allowing for specific imunoprecipitation of both MEF2C and the proteins MEF2C interacts with.

Detailed description

These biallelic mutant mice possess a FLAG/bio epitope tag downstream of the 3' UTR of Mef2c.

MEF2C (myocyte enhancer factor 2C) is a member of the MADS box family of transcription enhancer factors. MEF2C has both trans-activating and DNA binding properties and functions in myogenesis, development of the anterior heart field, neural crest and craniofacial development, and neurogenesis.

These mice also contain the Gt(ROSA)26Sor^{tm1.1(birA)Mejr} allele, which expresses HA (hemagglutinin) tagged bacterial birA, bifunctional protein (biotin ligase). In the homozygous state, Mef2c^tm1.1Wtp is perinatal lethal.

In these double mutant mice, MEF2C is biotinylated, allowing for its specific imunoprecipitation as well as any bound proteins.

Development

A targeting vector was designed to insert a FLAG/bio epitope tag and a frt-flanked neomycin resistance (neo) cassette at the endogenous Mef2c stop codon and upstream of the 3' UTR.

The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells and correctly targeted ES cells were injected into blastocysts. The resulting chimeric mice were bred to outbred Swiss Webster mice. Offspring were bred with Tg(ACTFLPe)9205Dym transgenic mice to delete the neo cassette. The resulting Mef2c-flbio mice were maintained on a Swiss Webster background.

The Gt(ROSA)26Sor^{tm1.1(birA)Mejr} allele was created using an HA (hemagglutinin) tagged bacterial birA, bifunctional protein (biotin ligase), gene inserted into exon 3 of the rabbit beta-globin gene. The entire birA ORF, with the rabbit beta globin splice acceptor and transcription termination sequences were excised to generate a targeting vector that also contained a floxed PGK- puromycin selection cassette. This targeting vector was inserted into the first intron of the Gt(ROSA)26Sor gene. The construct was electroporated into 129P2/OlaHsd derived IB10 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to FVB/N mice. The mice were intercrossed for 3 generations before being crossed to a Cre recombinase expressing strain (on an unknown genetic background) to remove the loxP-flanked PGK-puromycin selection cassette. A single loxP site remains. The Cre transgene was removed by breeding.

Mice carrying both alleles mice were maintained by the Cardiovascular Development Consortium (CvDC), an NIH-funded consortium within the Bench-to-Bassinett (B2B) program.

Upon arrival, mice were bred to FVB/NJ inbred mice for at least one generation to establish the colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(birA)Mejr}
Name: targeted mutation 1.1, Dies Meijer
MGI accession ID: MGI:3588507
Generation method: Targeted
Attributes: Not Applicable
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: 129P2/OlaHsd
Expressed genes: birA,biotin ligase),bacteria
Molecular note: An HA (hemagglutinin) tagged E. coli bacterial birA, bifunctional protein (biotin ligase), gene was inserted into exon 3 of the rabbit beta-globin gene. The entire birA ORF, with the rabbit beta globin splice acceptor and transcription termination sequences were excised to generate a targeting vector that also contained a floxed PGK- puromycin selection cassette. This targeting vector was inserted into the first intron of the Gt(ROSA)26Sor gene. Cre-mediated recombination removed the floxed puromycin cassette. Western blot of mutants demonstrated the expression of birA in all tissues tested, though levels of expression varied from tissue to tissue.

Allele

Symbol: Mef2c^tm1.1Wtp
Name: targeted mutation 1.1, William T Pu
MGI accession ID: MGI:5606040
Generation method: Targeted
Attributes: Inserted expressed sequence
Marker symbol: Mef2c
Marker name: myocyte enhancer factor 2C
Chromosome: 13
Strain of origin: 129S4/SvJae
Expressed genes: FLAG, FLAG octapeptide,
Molecular note: A targeting vector was designed to insert a FLAG/bio epitope tag and a frt-flanked neomycin resistance (neo) cassette at the endogenous Mef2c stop codon and upstream of the 3' UTR. FLP mediated recombination removed the FRT-flanked neo cassette.