STOCK Mir290a^{em1.1(Snrpn-tdTomato)Jae}/J

Stock number: 034183
Product name: M2T , Mir290-SE-RGM-tdTomato , Mir290-129^{SE-RGM-tdTomato} , M2TT , Mir290-SE-TT
Research areas: Cell Biology Research, Research Tools

Description

M2TT mice have a Snrpn::tdTomato::WPRE::BHGpA cassette inserted into the super enhancer (SE) sequences ~15 kb proximal of the Mir290a locus on the 129-derived chromosome 7.
The M2TT mice (Stock No. 034183) are designed for Reporter of Genome Methylation (RGM) studies via crossing to the M2GG mouse line homozygous for a Snrpn::eGFP::WPRE::BHGpA cassette in the Mir290a SE locus on the CAST-derived chromosome 7 (Stock No. 034184). Together, these allow for robustly tracking locus-specific DNA methylation at the Mir290a SE region and for functionally dissecting the hierarchy of epigenetic events that regulate enhancer activity and cellular states.

Detailed description

M2T is a CRISPR/cas9-made knock-in allele with a Snrpn::tdTomato::WPRE::BHGpA cassette inserted into the super enhancer (SE) sequences ~15 kb proximal of the Mir290a locus on the 129-derived chromosome 7. Heterozygous mice are called M2T, Mir290-SE-RGM-tdTomato or Mir290-129^{SE-RGM-tdTomato}, whereas homozygous mice are called M2TT or Mir290-SE-TT. Both heterozygous and homozygous mice are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities. Additionally, the donating investigator did not observe any defects in pre-implantation embryos - indicating that endogenous Mir290a expression is not adversely affected.

The Reporter of Genome Methylation (RGM) approach allows tracing of locus-specific DNA methylation based on the on-and-off of a fluorescent signal in single cells in real time, and has been shown to faithfully reflect the endogenous DNA methylation states. M2TT mice (Stock No. 034183) are designed for RGM studies via crossing to the M2GG mouse line homozygous for a Snrpn::eGFP::WPRE::BHGpA cassette in the Mir290a SE locus on the CAST-derived chromosome 7 (Stock No. 034184). This pluripotency SE overlaps with tissue-specific differential methylation regions (T-DMRs), which are hypo-methylated in ESCs but become de novo methylated upon differentiation.
Specifically, breeding M2GG and M2TT together results in compound heterozygous mice M2TG (also called Mir290-SE-TG or Mir290-129^{SE-RGM-tdTomato}/Mir290-CAST^SE-RGM-eGFP). Those M2TG animals allow direct fluorescent visualization of the Mir290 SE locus-specific DNA methylation state at allelic and single-cell resolution. Furthermore, M2TG enable dissection of allelic functional output of SE methylation states by distinguishing the two alleles based on the abundance of 129 or CAST allele-specific SNPs at both the DNA and the mRNA level. M2TG mice have tdTomato and eGFP fluorescence from 2-cell to E4.5 cells. From 4-cell stage onwards, the fluorescence is strong. [Song et al. 2019 Mol Cell 75:905 (PMID:31422875)]
Of note, the donating investigator reports that although the individual mutant mouse lines (M2TT or M2GG) exhibit different degrees of direct fluorescence among single cells, that observation could be due to allelic difference of methylation or could be due to transcriptional variation among cells independent of methylation (cell size, cell fate, etc). As such, they recommend RGM studies of the Mir290a SE use the compound heterozygous mice (M2TG) rather than the individual mouse lines (M2TT or M2GG).

Development

The M2T allele (called M2T, Mir290-SE-RGM-tdTomato or Mir290-129^{SE-RGM-tdTomato} as heterozygous and called M2TT or Mir290-SE-TT as homozygous) was created by the laboratory of Dr. Rudolf Jaenisch (Whitehead Institute, Massachusetts Institute of Technology) using CRISPR/cas9 endonuclease-mediated genome editing. This utilized oligonucleotides designed to target the super enhancer (SE) sequences ~15 kb proximal of the microRNA 290a locus (Mir290a) on chromosome 7, as well as Snrpn::tdTomato::WPRE::BHGpA::loxP-PGK-Puro-loxP sequences (encoding the minimal Snrpn promoter [including conserved elements between human and mouse and containing the endogenous imprinted DMR region], the tdTomato fluorescent protein, a WPRE sequence [to enhance the mRNA transcript stability], a BGH polyadenylation sequence and a loxP-flanked PGK-Puro selection cassette). The M2T allele was targeted into (129xCAST)F1 male embryonic stem (ES) cells as well as (C57BL/6 x 129S4/SvJae)F1 male-derived v6.5 ES cells, and the results pooled together. Junction PCR-SNP analyses identified ES cells with the M2T sequences (Snrpn::tdTomato::WPRE::BHGpA) inserted into the Mir290a SE on the 129 chromosome. Those ES cells were transfected with a Cre recombinase-expressing plasmid to remove the PGK-Puro, and then microinjected into recipient blastocysts. Chimeric males were bred to females on a C57BL/6J or (C57BL/6N x DBA/2)F1 genetic background. The resulting offspring were used establish the M2T colony. Thereafter, the M2T colony was maintained by breeding to C57BL/6 and/or breeding M2T mutant mice together to make homozygotes (M2TT). The donating investigator reports they screened their colony by internal probe (tdTomato Southern blot) to confirm it's single copy insertion at the correct locus, but the mice were not characterized for genome-wide off-target mutations. In 2020, homozygous males (black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Mir290a^{em1.1(Snrpn-tdTomato)Jae}
Name: endonuclease-mediated mutation 1.1, Rudolf Jaenisch
MGI accession ID: MGI:6383984
Generation method: Endonuclease-mediated
Attributes: Reporter
Synonyms: MIR290-129^{SE-RGM-tdTomato}; Mir290-SE-T
Marker symbol: Mir290a
Marker name: microRNA 290a
Marker synonyms: Mirn290; mmu-mir-290; mmu-mir-290a
Chromosome: 7
Strain of origin: 129
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing is used to target the super enhancer (SE) sequences ~15 kb proximal of the microRNA 290a locus on chromosome 7, as well as Snrpn::tdTomato::WPRE::BHGpA::loxP-PGK-Puro-loxP sequences (encoding the minimal Snrpn promoter, the tdTomato fluorescent protein, a WPRE sequence, a BGH polyadenylation sequence and a loxP-flanked PGK-Puro selection cassette). Junction PCR-SNP analyses identified ES c ells with the Snrpn::tdTomato::WPRE::BHGpA sequences inserted into the Mir290a SE on the 129S1 chromosome. Those ES cells were transfected with a Cre recombinase-expressing plasmid to remove the PGK-Puro cassette.