The R26R-Confetti conditional allele has a CAG promoter followed by a floxed-STOP cassette and the Brainbow 2.1 construct all targeted into the Gt(ROSA)26Sor locus. The R26R-Confetti allele functions as a stochastic multicolor Cre recombinase reporter of multiple fluorescent proteins from a single genomic locus. These R26R-Confetti mice allow a way to label and distinguish individual / adjacent cells with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in cre recombined cells.
Hans Clevers, Hubrecht Institute
Mice homozygous for the R26R-Confetti conditional allele are viable and fertile, with a CAG promoter, loxP site, and STOP cassette preventing transcription of the downstream Brainbow 2.1 sequences. The Brainbow 2.1 region contains two loxP-flanked dimers, each uniquely positioned in head-to-tail tandem. One dimer has nuclear-localized green fluorescent protein (hrGFPII) and a reverse-oriented cytoplasmic yellow fluorescent protein (mYFP). The other dimer has cytoplasmic red fluorescent protein (tdimer2(12)) and a reverse-oriented membrane-tethered cyan fluorescent protein (mCerulean). The Brainbow2.1 region may be written as loxP-STOP-loxP-GFP-PFY-Pxol-loxP-RFP-PFC-Pxol to show the transcriptional direction of each part. When bred to mice that express Cre recombinase, the resulting offspring may have a recombination event that stochastically places one of the four fluorescent proteins into position directly downstream of the CAG promoter within the cre-expressing tissues. Because this CAG promoter-driven Brainbow 2.1 reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, fluorescent protein expression is determined by which tissues express Cre recombinase. The donating investigator reports that mice do not express any fluorescent cells prior to introduction of Cre recombinase. The donating investigator confirms fluorescent protein expression following exposure to cre can be detected by direct fluorescence (and presumably also via mRNA (in situ hybridization) and antibody staining (immunohistochemistry)).
Initial Cre recombination outcomes may recombine the loxP-flanked STOP cassette (green), invert the loxP-STOP-loxP-GFP-PFY-Pxol region (yellow), recombine the loxP-STOP-loxP-GFP-PFY-Pxol-loxP region (red), or invert the entire loxP-STOP-loxP-GFP-PFY-Pxol-loxP-RFP-PFC-Pxol region (blue). Other recombination outcomes may not remove the STOP cassette and result in no fluorescent reporter labeling in cre-expressing cells. In addition, sequential recombination outcomes may reduce the construct to a single invertible dimer segment that can continue to invert as long as Cre recombinase is present. The donating investigator also reports that weaker cre expression favors inverting the loxP-STOP-loxP-GFP-PFY-Pxol region rather than removal of the loxP-STOP-loxP region: this results in less green-fluorescing cells / more non-fluorescing cells than is expected if using a strong cre-expressing line.
A targeting vector containing (from 5' to 3') a strong CAGG promoter (CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter), a loxP site, a PGK-Neor-pA cassette (serving as a transcriptional roadblock), and the Brainbow 2.1 construct (described in greater detail below). This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into 129P2/OlaHsd-derived IB10/E14IB10 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric males were bred with C57BL/6 females to generate the R26R-Confetti colony. The resulting R26R-Confetti colony were bred with other mutant or cre-expressing mice, but these other mutations were bred away from the R26R-Confetti colony. The R26R-Confetti mice are reported to be on a genetic background equivalent to ~2-3 backcross generations to C57BL/6 prior to sending to The Jackson Laboratory Repository in 2010. Upon arrival the mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
The Brainbow 2.1 construct was designed by Drs. Jeff Lichtman and Joshua Sanes (Harvard University) with four fluorescent protein sequences uniquely positioned in a tandem fashion and delimited by loxP sites in opposite orientation. Specifically, this Brainbow 2.1 coding region is composed of two adjacent floxed head-to-tail tandem dimers. The first head-to-tail dimer contains a loxP site and humanized Renilla GFP (hrGFPII; with nuclear localization signal plus polyA sequence) in forward orientation, and a loxP site and monomeric EYFP (mYFPA206K plus polyA sequence) in reverse orientation. The second head-to-tail dimer contains a loxP site and tdimer2(12) RFP plus polyA sequence in forward orientation, and a loxP site and mCerulean CFP (with membrane tethering palmitoylation sequence plus polyA sequence) in reverse orientation. A single frt site is located at the 3' end of the Brainbow 2.1 construct. The entire construct may be written as loxP-STOP-loxP-GFP-PFY-Pxol-loxP-RFP-PFC-Pxol-frt to show transcriptional direction of each part.
The hrGFPII variant of GFP (from Stratagene vector phrGFPII-C) has amino acid substitutions designed to improve spectral properties and performance in mammalian systems. The monomeric EYFP (mYFPA206K) has an amino acid substitution replacing a hydrophobic region with a positively charged residue designed to prevent dimerization. The tdimer2(12) RFP is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer). The monomeric Cerulean (mCerulean) is a variant of ECFP (ECFPS72A/Y145A/H148D/A206K) with amino acid substitutions designed to improve spectral properties and prevent dimerization.
|Allele Name||targeted mutation 1, Hans Clevers|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Reporter)|
|Allele Synonym(s)||R26R-Brainbow2.1; R26R-Confetti|
|Gene Symbol and Name||Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano|
|Gene Synonym(s)||AV258896; Gtrgeo26; Gtrgeo26; Gtrosa26; Gtrosa26; R26; ROSA26; SETD5-AS1; beta geo; expressed sequence AV258896; gene trap ROSA 26; gene trap ROSA b-geo 26|
|Site of Expression||Cre recombination results in stochastic multicolor fluorescent proteins being expressed from a single genomic locus in cre-expressing tissues.|
|Strain of Origin||129P2/OlaHsd|
|Mutations Made By|| |
Hans Clevers, Hubrecht Institute
When maintaining a live colony, heterozygous mice may be bred together or with wildtype mice from the colony. The donating investigator reports breeding homozygous mice together with no fertility or viability problems.
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