B6.FVB-Tg(CAG-EGFP,-Sumo1,-Sumo2,-Sumo3,-mCherry)10Weiy/J

Stock number: 031693
Product name: CAG-SUMO line 10
Research areas: Cancer Research, Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

CAG-SUMO line 10 mice have widespread EGFP fluorescence that can be replaced by SUMO1/2/3 paralog expression and mCherry fluorescence after Cre recombinase exposure. These mice may be useful for studying in vivo SUMO proteomics - for example, mechanistically linking SUMO conjugation (SUMOylation) and the SUMO-modified proteome to the pathological processes of human disorders.

Detailed description

SUMOylation is the process by which small ubiquitin-like modifiers (SUMOs) are covalently conjugated to lysine residues of target proteins - thereby modulating their function, stability and localization. SUMO conjugation modulates many cellular functions including DNA repair, genome maintenance, gene transcription and protein degradation control, and plays key roles in many human diseases such as cerebral ischemia/stroke, cancer and heart failure.

CAG-SUMO line 10 mice have the CAG promoter/enhancer and a floxed-EGFP-STOP cassette that prevents expression of a downstream polycistronic construct encoding three individual N-terminal marker-tagged mouse SUMO precursors (His-SUMO1, HA-SUMO2 and FLAG-SUMO3) and the mCherry fluorescent reporter - each separated by 2A oligopeptide sequences.

In the absence of Cre recombinase, the donating investigator reports widespread EGFP expression (generally dim fluorescence) is observed in all organs examined (including brain, heart, lung, kidney and liver), and there is no transgene expression of the N-terminal-tagged SUMO1/2/3 paralogs or mCherry. They also report CAG-SUMO line 10 has retained a consistent pattern of EGFP expression through several generations of backcrossing onto C57BL/6J. Hemizygous CAG-SUMO line 10 mice are viable and fertile with no developmental, behavioral or reproductive abnormalities. It has not been attempted to make this strain homozygous to date (March 2018).

Following Cre-mediated deletion of the floxed-EGFP-STOP cassette, expression of the N-terminal-tagged SUMO1/2/3 paralogs (His-SUMO1, HA-SUMO2 and FLAG-SUMO3) is observed along with robust mCherry fluorescence. The endogenous SUMO proteases remove the extra 2A peptides and expose the C-terminal di-glycine motif of each SUMO. All three N-terminal-tagged SUMO1/2/3 paralogs are functionally active, and their expression did not modify the endogenous SUMOylation machinery - that is, these three exogenous SUMOs are processed by the endogenous SUMOylation machinery in the same way as endogenous SUMOs.
For example, when CAG-SUMO line 10 are bred to Emx1-IRES-Cre mice (Stock No. 005628), the resulting CAG-SUMO/Emx1-Cre mice showed strong mCherry expression restricted to forebrain regions (cerebral cortex and hippocampus), and retained EGFP fluorescence in widespread other tissues. Such CAG-SUMO/Emx1-Cre mice are useful for studying the SUMO-modified proteome regulated by a stress condition in the brain (such as ischemia).

Development

The CAG-SUMO transgenic mouse line 10 was created by Dr. Wei Yang (Duke University Medical Center).

The CAG-SUMO transgene was designed to have (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP site, an enhanced green fluorescent protein gene (EGFP), a transcriptional/translational STOP cassette (containing a PGK-neo), a second loxP site, a mouse cDNA sequence encoding precursor SUMO1 with N-terminal histidine tag (His-SUMO1), a viral 2A oligopeptide sequence (P2A; mediates ribosomal skipping), a mouse cDNA sequence encoding precursor SUMO2 with N-terminal hemagglutinin tag (HA-SUMO2), a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), a mouse cDNA sequence encoding precursor SUMO3 with N-terminal FLAG tag (FLAG-SUMO3), a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), the monomeric DsRed fluorescent protein variant mCherry gene and a bovine growth hormone polyadenylation signal.
The ~9.4 kbp transgene was injected into fertilized FVB/N oocytes, and transgenic founders were bred with C57BL/6J mice for germline expression. Founder line 10 was found to have widespread EGFP expression, and was further characterized. The CAG-SUMO line 10 colony was backcrossed to C57BL/6J inbred mice for more than ten generations, and then hemizygous males with black coat color were sent to The Jackson Laboratory Repository in 2018. 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).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). In addition, the transgene insertion site(s) and copy number are not known to date (March 2018).

Allele

Symbol: Tg(CAG-EGFP,-Sumo1,-Sumo2,-Sumo3,-mCherry)10Weiy
Name: transgene insertion 10, Wei Yang
MGI accession ID: MGI:6150883
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence; Epitope tag
Synonyms: CAG-SUMO
Marker symbol: Tg(CAG-EGFP,-Sumo)10Weiy
Marker name: transgene insertion 10, Wei Yang
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: In the absence of Cre recombinase, the donating investigator reports widespread, but dim, EGFP expression is observed in all organs examined (including brain, heart, lung, kidney and liver). Following Cre-mediated deletion of the floxed-EGFP-STOP cassette, robust mCherry fluorescence is seen in Cre-expressing tissues.
Molecular note: The transgene was designed to have (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP site, EGFP, a transcriptional/translational STOP cassette (containing a PGK-neo), a second loxP site, a mouse cDNA sequence encoding precursor SUMO1 with N-terminal histidine tag (His-SUMO1), a viral 2A oligopeptide sequence (P2A; mediates ribosomal skipping), a mouse cDNA sequence encoding precursor SUMO2 with N-terminal hemagglutinin tag (HA-SUMO2), a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), a mouse cDNA sequence encoding precursor SUMO3 with N-terminal FLAG tag (FLAG-SUMO3), a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), the monomeric DsRed fluorescent protein variant mCherry gene and a bovine growth hormone polyadenylation signal. Eight founder lines were created, founder line 10 exhibits ubiquitous expression of GFP.