STOCK Gt(ROSA)26Sor^{tm1(CAG-EYFP*,-mEYFP*,-tdTomato*,-mTFP1*)Ben}/Gt(ROSA)26Sor^{tm2(CAG-EYFP*,-mCherry*,-EGFP*,-mCerulean*)Ben}/J

Stock number: 031298
Product name: iMb2-Control-Mosaic::iChr2-Control-Mosaic
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous mice allow several possible Cre recombination outcomes in any single cell. Each outcome results in expression of both a membrane-bound fluorescent protein and a chromatin/nuclear-localized fluorescent protein, and each is mutually exclusive and permanent. These mice are also designed to permit further introduction of genes of interest for inducible, fluorescent and functional genetic mosaic analysis (ifgMosaic or Dual ifgMosaic).
Mice are also available with each individual allele: iMb2-Control-Mosaic (Stock No. 031301) and iChr2-Control-Mosaic (Stock No. 031302).

Detailed description

iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous mice have the iMb2-Control-Mosaic allele in the Gt(ROSA)26Sor locus on one homologous chromosome and the iChr2-Control-Mosaic allele in the Gt(ROSA)26Sor locus on the other homologous chromosome.
Both alleles have three fluorescent protein sequences - each uniquely flanked with pairs of incompatible lox sites (in the same direction). The fluorescent proteins are targeted to the membrane or the chromatin/nucleus in iMb2-Control-Mosaic or iChr2-Control-Mosaic, respectively. Each allele has three possible recombination outcomes that are mutually exclusive and permanent in any single cell.
Prior to Cre recombinase exposure, the donating investigator reports that compound heterozygous mice are viable and fertile with normal breeding, and exhibit no behavioral, neurological or any other spontaneous abnormalities. Each allele has widespread expression of the nuclear-localized, non-cytotoxic/non-fluorescent protein N-PhiMut, and does not express any of its three downstream fluorescent proteins.
Following Cre recombinase exposure, compound heterozygotes have the N-PhiMut sequences deleted and then each allele has one of its three downstream fluorescent proteins placed into the proper orientation for expression (see below) - resulting in single cells with simultaneous expression of both a membrane-bound fluorescent protein and a chromatin-localized fluorescent protein. This can generate up to nine different cell clones with dual labeling of the membrane (for cell shape) and chromatin/nuclei (for cell unit count and proliferation analysis).
Specifically, the iMb2-Control-Mosaic allele recombines to express either (i) a membrane-targeted monomeric EYFP (Mb2-mEYFP), (ii) a membrane-targeted tdTomato (Mb2-tdTomato) or (iii) a membrane-targeted monomeric teal fluorescent protein with N-terminal hemagglutinin tag (Mb2-HA-mTfp1). The iChr2-Control-Mosaic allele recombines to express either (i) a chromatin/nuclear-targeted mCherry with N-terminal 6xHis affinity tag (6xHis-H2B-mCherry), (ii) a chromatin/nuclear-targeted EGFP (H2B-EGFP) or (iii) a chromatin/nuclear-targeted mCerulean with N-terminal hemagglutinin tag (HA-H2B-mCerulean). The donating investigator reports that each fluorescent protein exhibits bright fluorescence once it is expressed. Additionally, they report that presence of the frt-flanked PGK/EM7-neo-BGHpA selection cassette (just upstream of the second 2xINS sequence) in each allele does not adversely affect expression (compared to the respective post-FLP allele without the PGK/EM7-neo-BGHpA selection cassette).

The donating investigator (Dr. Rui Benedito) has made mouse lines available with the individual alleles, iMb2-Control-Mosaic (Stock No. 031301) and iChr2-Control-Mosaic (Stock No. 031302), as well as the iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygotes (Stock No. 031298). A diagram showing the iMb2-Control-Mosaic and iChr2-Control-Mosaic design, as well as their recombination possibilities, was assembled in collaboration with Dr. Benedito and is available here. Of note, the diagram does not show the frt-flanked PGK/EM7-neo-BGHpA selection cassette that is just upstream of the second 2xINS sequence in each allele.

As described in the publication characterizing these mice [Pontes-Quero et al. 2017 Cell 170:800], iMb2-Control-Mosaic::iChr2-Control-Mosaic mice can be used for Dual ifgMosaic analysis - allowing multiplex dual labeling of the membrane and chromatin in recombined cells for higher-resolution analysis of the clone-cell number and shape in pulse and chase genetic experiments. A total of 15 distinct cell clones can be induced in a given tissue expressing Cre or CreERT2. Six of these clones will have the expression of only one fluorescent protein, and the other nine clones will express a distinct combination of two different proteins - one localized at the membrane and the other in the nuclei. When these mice are combined with other floxed alleles, they also allow one to accurately track the proliferation and migration of individual mutant cells with Cre activity. If one of these Control-Mosaic alleles is combined with other ifgMosaic alleles containing functional genes, then clonal genetic and cellular resolution is increased. iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous mice can also be used as controls of combinatorial genetic epistasis analysis experiments that are possible to perform after intercrossing two ifgMosaic functional alleles. In this case, the function of three genes can be studied in the context of any other three expressed genes, in the same tissue, and in a total of nine different combinations (3x3=9).
Regarding the overall Cre-mediated recombination frequencies for the iMb2-Control-Mosaic and iChr2-Control-Mosaic alleles, in general, there is an inverse correlation between the recombination outcome and the genetic distance separating the compatible lox sites. That is, weaker Cre expression may bias recombination to the easiest possibility (the first fluorescent protein). The recombination frequencies may also depend also on other factors. For example, stronger tissue-specific Cre expression may lead to more equal outcome frequencies. Several examples are described in the publication characterizing these mice [Pontes-Quero et al. 2017 Cell 170:800].

Development

The iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous mice were designed by Dr. Rui Benedito (Centro Nacional de Investigaciones Cardiovasculares [CNIC]). These mice have the iMb2-Control-Mosaic allele in the Gt(ROSA)26Sor locus on one homologous chromosome and the iChr2-Control-Mosaic allele in the Gt(ROSA)26Sor locus on the other homologous chromosome.
The iMb2-Control-Mosaic allele has three membrane-targeted fluorescent protein sequences uniquely flanked with pairs of incompatible lox sites (in the same direction) - see Stock No. 031301 for details. The iChr2-Control-Mosaic allele has three chromatin/nuclear-targeted fluorescent protein sequences uniquely flanked with pairs of incompatible lox sites (in the same direction) - see Stock No. 031302 for details.
To generate the iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous mice, Dr. Benedito bred the two mutant lines together. At some point, they were bred with C57BL/6N-congenic animals with non-germline CreERT2 expression; the Cdh5-CreERT2 transgene was later removed. In 2018, iMb2-Control-Mosaic::iChr2-Control-Mosaic compound heterozygous males on a mixed C57BL/6;129S6 genetic background were sent to The Jackson Laboratory as Stock No. 031298. Upon arrival, they were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish our live colony.

A diagram showing the iMb2-Control-Mosaic and iChr2-Control-Mosaic design, as well as their recombination possibilities, was assembled in collaboration with Dr. Benedito and is available here. Of note, the diagram does not show the frt-flanked PGK/EM7-neo-BGHpA selection cassette that is just upstream of the second 2xINS sequence in each allele.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-EYFP*,-mEYFP*,-tdTomato*,-mTFP1*)Ben}
Name: targeted mutation 1, Rui Benedito
MGI accession ID: MGI:6108157
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: iMb2-Control-Mosaic
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: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral; CFP,Cyan Fluorescent Protein,jellyfish; GFP,Green Fluorescent Protein,; YFP,Yellow Fluorescent Protein,jellyfish; HA tag,HA tag,
Site of expression: Fluorescent proteins will be expressed in in tissues expressing Cre recombinase driven by a selected promoter.
Molecular note: This allele has three membrane-targeted fluorescent protein sequences - each uniquely flanked with pairs of incompatible lox sites (in the same direction) inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus with a CAG hybrid promoter/enhancer. The targeting vector is designed with (from 5' to 3') two copies of chicken beta-globin HS4 insulator element (2xINS; increase downstream gene expression and minimize regulatory interference), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxN site, a lox2272 site, a loxP site, the non-cytotoxic/non-fluorescent Phialidium yellow fluorescent protein variant PhiYFP with nuclear localization signal (N-PhiMut), SV40 polyA signal sequence, a second loxN site, a full consensus KOZAK sequence, the Mb2-YFP gene (monomeric enhanced yellow fluorescent protein [mYFP^A206K] with N-terminal 41 amino acid membrane-targeting tag sequence), in-frame Thosea asigna virus 2A cleavage site (T2A; mediates ribosomal skipping), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), SV40 polyA signal sequence, a second lox2272 site, a full consensus KOZAK sequence, the Mb2-Tomato gene (non-oligomerizing tandem-dimer DsRed fluorescent protein variant tdTomato with N-terminal 41 amino acid membrane-targeting tag sequence), in-frame T2A sequence, WPRE sequence, SV40 polyA signal sequence, a second loxP site, a full consensus KOZAK sequence, the Mb2-HA-TFP1 gene (monomeric teal fluorescent protein mTFP1 with N-terminal hemagglutinin tag and 41 amino acid membrane-targeting tag sequence), in-frame T2A sequence, WPRE sequence, SV40 polyA signal sequence, an FRT site flanked neomycin resistance gene cassette, and a second 2xINS sequence.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-EYFP*,-mCherry*,-EGFP*,-mCerulean*)Ben}
Name: targeted mutation 2, Rui Benedito
MGI accession ID: MGI:6108166
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Epitope tag
Synonyms: Gt(Rosa)26Sor^{tm1(iChr2-Control-Mosaic)}; iChr2-Control-Mosaic
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: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral; YFP,Yellow Fluorescent Protein,jellyfish; CFP,Cyan Fluorescent Protein,jellyfish; HA tag,HA tag,
Site of expression: Fluorescent proteins will be expressed in in tissues expressing Cre recombinase driven by a selected promoter.
Molecular note: This allele has three chromatin/nuclear-targeted fluorescent protein sequences uniquely flanked with pairs of incompatible lox sites (in the same direction), all inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. The targeting vector is designed with (from 5' to 3') two copies of chicken beta-globin HS4 insulator element (2xINS; increase downstream gene expression and minimize regulatory interference), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxN site, a lox2272 site, a loxP site, the N-PhiMut gene (non-cytotoxic/non-fluorescent Phialidium yellow fluorescent protein variant PhiYFP with nuclear localization signal), SV40 polyA signal sequence, a second loxN site, a full consensus KOZAK sequence, the 6xHis-H2B-mCherry gene (monomeric DsRed fluorescent protein variant mCherry with N-terminal 6xHis affinity tag and 125 amino acid human histone 2B chromatin/nucleus tag), in-frame Thosea asigna virus 2A cleavage site (T2A; mediates ribosomal skipping), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), SV40 polyA signal sequence, a second lox2272 site, a full consensus KOZAK sequence, the H2B-EGFP gene (enhanced green fluorescent protein with 127 amino acid human histone 2B chromatin/nucleus tag and KOZAK sequence plus C-terminal paramyxovirus V5 tag sequence), in-frame T2A sequence, WPRE sequence, SV40 polyA signal sequence, a second loxP site, a full consensus KOZAK sequence, the HA-H2B-mCerulean gene (monomeric enhanced cyan fluorescent protein variant mCerulean with N-terminal hemagglutinin (HA) tag, 125 amino acid human histone 2B chromatin/nucleus tag and KOZAK sequence), in-frame T2A sequence, WPRE sequence, SV40 polyA signal sequence, an FRT site flanked neomycin resistance gene cassette, and a second 2xINS sequence.