B6.129-Gt(ROSA)26Sor^{tm3(CAG-EGFP/Dsred2)Luo}/J

Stock number: 006075
Product name: MADM-GR
Research areas: Cell Biology Research, Research Tools

Description

These knock-in mice were developed to utilize the mosaic analysis with double markers (MADM) system that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. This strain carries the green fluorescent protein, EGFP (G), and the red fluorescent protein, Dsred2 (R), as markers to be used in conjunction with strains carrying the reciprocal chimeric marker genes (MADM-RG).

Detailed description

MADM-GR mice are viable with no gross behavioral or observable abnormalities. Homozygous mice have low fertility, while heterozygous mice have no reported fertility defects. These mutants are designed for MADM (mosaic analysis with double markers), and must be crossed to mice harboring a reciprocal mutation at the same locus (see Stock No. 006067 or Stock No. 006080, MADM-RG (Dsred2/EGFP)). The resulting offspring have one copy of each reciprocal mutation on homologous chromosomes ("trans-heterozygous") and must next be bred to a Cre-expressing strain for fluorescent protein expression. Prior to Cre-recombination, double mutant mice do not have colored cells: the chimeric genes do not produce functional proteins because their coding sequences are interrupted by the beta-actin intron in different reading frames. After DNA replication (G2 phase) in double mutant mice, Cre-recombinase introduction that facilitates inter-chromosomal recombination aligns the respective N- and C-terminal coding sequences for each of the reporter genes on the same chromosome. The following chromatid segregation (X or Z) determines daughter cell phenotype (recombinant sister chromatids into the same daughter cell leads to double reporter expression [a G2-Z event], while independent segregation into separate daughter cells leads to expression of EGFP or Dsred2-MYC [a G2-X event]). If an additional targeted mutation of interest is introduced distal to the Gt(ROSA) locus, only homozygous cells will be singly labeled following G2 Cre-introduction. The homozygous mutant and wild type cells can then be distinguished by which single reporter they express. Reporter protein tissue specificity, expression levels, and frequency of recombination are thus determined by the promoter controlling Cre expression. Using this MADM system, a researcher can generate genetic mosaics in which an individual organism contains somatic cells of different genotypes. This allows the researcher to ascertain lineal relationships and pleiotropic gene function in multicellular organisms. These mice may also be useful in studies of cell differentiation and mitosis.

Mice harboring this MADM-GR mutation are also available on their original 129 genetic background (see Stock No. 006041). A control strain (Stock No. 006071, MADM-GG (EGFP/EGFP)) is also provided in which EGFP is in-frame on the same chromosome. Other important features of the MADM system are listed below. Cre-recombinase introduction in cell phase G0 or G1 results in double reporter expression. While EGFP expression can be visualized in vivo and in fixed samples, Dsred2-MYC can not. Anti-MYC immunofluorescence allows simultaneous visualization of both reporter genes. Single copy Dsred2-MYC is inadequate for live visualization.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

Development

A targeting vector was designed to contain the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; Okada et al, Exp Neurol 1999 156:394-406), beta-globin intronic sequence (itself containing a loxP-flanked neomycin resistance gene), and C-terminal portion of a red fluorescent protein (Dsred2; Clonetech) tagged with six copies of the MYC epitope at its C-terminus. This construct (GR) is preceded by the cytomegalovirus beta-actin enhancer-promoter and followed by the SV40 T antigen poly A signal, allowing for ubiquitous and high-level expression of the reporter genes. This entire construct was inserted into the Gt(ROSA)26Sor locus via electroporation of (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were microinjected in C57BL/6 blastocysts and chimeric progeny were established. Mutant mice were bred to 129S1/SvImJ (Stock No. 002448) for at least three generations, and then intercrossed to homozygosity before arriving at The Jackson Laboratory (as Stock No. 006041). Upon arrival, mice were backcrossed to C57BL/6J for at least 5 generations to establish this colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm3(CAG-EGFP/Dsred2)Luo}
Name: targeted mutation 3, Liqun Luo
MGI accession ID: MGI:3623347
Generation method: Targeted
Attributes: Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: when crossed to reciprocal strain (Stock No. 006067) and then to a cre strain, differential expression of the reporter protein(s) will be expressed in daughter cells, depending on chromatid segregation
Molecular note: A construct containing the CMV beta-actin enhancer/promoter, SV40 poly(A) signal, and encoding the N-terminal part of EGFP and C-terminal part of Dsred2 (red fluorescent protein; also tagged with 6 copies of the MYC epitope) separated by a beta-globin intron containing a loxP-flanked neomycin resistance gene, was knocked into ES cells at the ROSA26 locus. No protein is expressed from the chimeric locus in absence of cre-mediated recombination because the coding sequences are interrupted by the intron in different reading frames. After recombination with the reciprocal mutation at the same locus carried by another mouse line, which restores the reading frames of EGFP and RFP, RNA splicing will remove the loxP sites, resulting in functional protein expression.