STOCK Sox2^{em2.1(Snrpn-EGFP)Jae}/J

Stock number: 034182
Product name: S2G , SOX2-SE-RGM-eGFP , SOX2-CAST^SE-RGM-eGFP , S2GG , SOX2-SE-GG
Research areas: Cell Biology Research, Research Tools

Description

S2GG mice have a Snrpn::eGFP::WPRE::BHGpA cassette inserted into the super enhancer (SE) sequences ~100 kb distal of the Sox2 locus on the CAST-derived chromosome 3.
The S2GG mice (Stock No. 034182) are designed for Reporter of Genome Methylation (RGM) studies via crossing to the S2TT mouse line homozygous for a Snrpn::tdTomato::WPRE::BHGpA cassette in the Sox2 SE locus on the 129-derived chromosome 3 (Stock No. 034181). Together, these allow for robustly tracking locus-specific DNA methylation at the Sox2 SE region and for functionally dissecting the hierarchy of epigenetic events that regulate enhancer activity and cellular states.

Detailed description

S2G is a CRISPR/cas9-made knock-in allele with a Snrpn::eGFP::WPRE::BHGpA cassette inserted into the super enhancer (SE) sequences ~100 kb distal of the Sox2 locus on the CAST-derived chromosome 3. Heterozygous mice are called S2G, SOX2-SE-RGM-eGFP or SOX2-CAST^SE-RGM-eGFP, whereas homozygous mice are called S2GG or SOX2-SE-GG. Both heterozygous and homozygous mice are viable and fertile with normal breeding, and no gross phenotypic abnormalities. The donating investigator reports some heterozygous or homozygous mice can exhibit mild hyperactivity - which could be due to the S2G targeted to the Sox2 allele from CAST (a strain documented as aggressive). Additionally, they did not observe any defects in pre-implantation homozygous embryos, nor in later stages - indicating that endogenous Sox2 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. S2GG mice (Stock No. 034182) are designed for RGM studies via crossing to the S2TT mouse line homozygous for a Snrpn::tdTomato::WPRE::BHGpA cassette in the Sox2 SE locus on the 129-derived chromosome 3 (Stock No. 034181). 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 S2TT and S2GG together results in compound heterozygous mice S2TG (also called SOX2-SE-TG or Sox2-129^{SE-RGM-tdTomato}/Sox2-CAST^SE-RGM-eGFP). Those S2TG animals allow direct fluorescent visualization of the Sox2 SE locus-specific DNA methylation state at allelic and single-cell resolution. Furthermore, S2TG 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. S2TG mice have tdTomato and eGFP fluorescence in morula-E4.5 cells that, from blastocyst onwards, should be confined in the inner cell mass. From late morula 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 (S2TT or S2GG) 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 Sox2 SE use the compound heterozygous mice (S2TG) rather than the individual mouse lines (S2TT or S2GG).

Development

The S2G allele (called S2G, SOX2-SE-RGM-eGFP or SOX2-CAST^SE-RGM-eGFP as heterozygous and called S2GG or SOX2-SE-GG as homozygous) was created by the laboratory of Dr. Rudolf Jaenisch (Whitehead Institute, Massachusetts Institute of Technology) using CRISPR/cas9 endonuclease-mediated genome editing in (129xCAST)F1 male embryonic stem (ES) cells. This utilized oligonucleotides designed to target the super enhancer (SE) sequences ~100 kb distal of the SRY (sex determining region Y)-box 2 locus (Sox2) on chromosome 3, as well as Snrpn::eGFP::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 enhanced green fluorescent protein (EGFP), a WPRE sequence [to enhance the mRNA transcript stability], a BGH polyadenylation sequence and a loxP-flanked PGK-Puro selection cassette). Junction PCR-SNP analyses identified ES cells with the S2G sequences (Snrpn::eGFP::WPRE::BHGpA) inserted into the Sox2 SE on the CAST 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 C57BL/6J females, and the resulting agouti offspring were used establish the S2G colony. The colony was maintained by breeding S2G mutant mice together to make homozygotes (S2GG). The donating investigator reports they screened their colony by internal probe (eGFP Southern blot) to confirm its single copy insertion at the correct locus, but the mice were not characterized for genome-wide off-target mutations. In 2020, homozygous males (agouti 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: Sox2^{em2.1(Snrpn-EGFP)Jae}
Name: endonuclease-mediated mutation 2.1, Rudolf Jaenisch
MGI accession ID: MGI:6383982
Generation method: Endonuclease-mediated
Attributes: Reporter
Synonyms: SOX2-CAST^SE-RGM-eGFP; SOX2-SE-G
Marker symbol: Sox2
Marker name: SRY (sex determining region Y)-box 2
Marker synonyms: ANOP3; lcc; MCOPS3; RGD1565646; Sox-2; ysb
Chromosome: 3
Strain of origin: CAST/Ei
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing in is used target the super enhancer (SE) sequences ~100 kb distal of the SRY (sex determining region Y)-box 2 locus (Sox2) on chromosome 3, as well as Snrpn::eGFP::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 enhanced green fluorescent protein (EGFP), a WPRE sequence, a BGH polyadenylation sequence and a loxP-flanked PGK-Puro selection cassette). Junction PCR-SNP analyses identified ES cells with Snrpn::eGFP::WPRE::BHGpA sequences inserted into the Sox2 SE on the CAST chromosome. Those ES cells were transfected with a Cre recombinase-expressing plasmid to remove the PGK-Puro cassette.