129(Cg)-Gt(ROSA)26Sor^{tm5(CAG-Sun1/sfGFP)Nat}/CdnyJ

Stock number: 039976
Product name: 129S.CAG-Sun1/sfGFP
Research areas: Cell Biology Research, Research Tools

Description

129S.CAG-Sun1/sfGFP mice, backcrossed to 129S, are used in the INTACT (isolation of nuclei tagged in specific cell types) method for immunopurification of nuclei. Cre-dependent removal of a floxed STOP cassette allows expression of the SUN1 fusion protein at the inner nuclear membrane in targeted cell types. These nuclei can be immunopurified with antibodies against GFP or MYC for transcriptional and epigenetic studies.

Note that this allele is also available on a mixed B6;129 background (Stock No. 021039) and on a C57BL/6-congenic background (Stock No. 030952).

Detailed description

129S.CAG-Sun1/sfGFP mice, backcrossed to 129S, are used in the INTACT (isolation of nuclei tagged in specific cell types) method for immunopurification of nuclei. These mice contain a CAG promoter driving expression of coding sequences for the mouse nuclear membrane protein SUN1 (Sad1 and UNC84 domain containing 1) fused at its C-terminus to 2 copies of superfolder GFP (sfGFP) followed by 6 copies of Myc, inserted in the Gt(ROSA)26Sor locus. Expression of Sun1-sfGFP-Myc is prevented by an upstream loxP-flanked STOP cassette. Cre-dependent removal of the floxed STOP cassette allows expression of the SUN1 fusion protein at the inner nuclear membrane in targeted cell types. These nuclei can be immunopurified with antibodies against GFP or MYC for transcriptional and epigenetic studies. Mice homozygous for this allele are viable and fertile.

Note that this allele is also available on a mixed B6;129 background (Stock No. 021039) and on a C57BL/6-congenic background (Stock No. 030952).

Development

The CAG-Sun1/sfGFP targeting vector, originally designed by Dr. Jeremy Nathans, contains (from 5' to 3') a CAG promoter, a loxP-flanked STOP cassette, a mouse Sun1 fused with 2 copies of superfolder green fluorescent protein (GFP) and 6 copies of Myc, and an Frt-flanked PGK-neomycin cassette. The entire targeting vector was inserted into the Gt(ROSA)26Sor gene. The construct was electroporated into 129-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. Resulting chimeric males were crossed to C57BL/6 mice and then intercrossed to homozygosity (Stock No. 021039).

Dr. Christine Ann Denny purchased some Stock No. 021039 animals, on the mixed genetic background, and backcrossed these to an 129/SvEV inbred mouse line for at least 9 generations before sending mice back to The Jackson Laboratory in 2025. The resulting 129S-congenic strain is called 129S.Cg-Gt(ROSA)26Sor^{tm5(CAG-Sun1/sfGFP)Nat}/CdnyJ (Stock No. 039976). To establish a live colony at The Jackson Laboratory, 129S.CAG-Sun1/sfGFP females and males were bred together and embryos cryopreserved.

Allele

Symbol: Gt(ROSA)26Sor^{tm5(CAG-Sun1/sfGFP)Nat}
Name: targeted mutation 5, Jeremy Nathans
MGI accession ID: MGI:5443817
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
Synonyms: R26-CAG-LSL-Sun1-sfGFP-Myc; SUN1-2xsfGFP-6xMYC; Sun1-LsL; CAG-Sun1sfGFP; Sun1^LsL
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: 129
Expressed genes: GFP,Green Fluorescent Protein,; Sun1,Sad1 and UNC84 domain containing 1,mouse, laboratory; Myc,myelocytomatosis oncogene,mouse, laboratory
Site of expression: The SUN1 fusion protein (SUN1 fused to sfGFP) is expressed at the inner nuclear membrane in targeted cell types where promoters driving Cre recombinase are expressed.
Molecular note: A targeting vector containing (from 5' to 3') a CAG promoter, a loxP site flanked STOP cassette, the mouse Sun1 cDNA fused with 2 copies of the superfolder GFP (sfGFP) green fluorescent protein gene and 6 copies of the Myc tag, and an FRT site flanked neomycin resistance gene cassette was inserted into the locus. Removal of the floxed stop cassette is required to achieve expression.