STOCK Gt(ROSA)26Sor^{tm7(ACTB-EGFP*)Luo}/J

Stock number: 017921
Product name: R26^TG
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

The R26^TG allele has a CMV enhancer/chicken beta-actin core promoter-driven "MADM TG" cassette (ATG-intron-GFP^C-terminus) inserted into the Gt(ROSA)26Sor locus on chromosome 6. The new TG cassette in R26^TG mice is compatible with any "GX cassette" (GFP^N-terminus-intron-X^ATG-less) where X^ATG-less is any gene without the start codon. Like the original MADM system, the "new MADM-6" system provides a tool to generate genetic mosaics in which an individual organism contains somatic cells of different genotypes. This allows Cre or FLP recombinase-induced fluorescent labeling of daughter cells to ascertain lineal relationships and pleiotropic gene function in multicellular organisms. These mice may also be useful in studies of cell differentiation and mitosis, as well as for the MADM-Tet system.

Detailed description

The R26^TG allele has a CMV enhancer/chicken beta-actin core promoter-driven "MADM TG" cassette inserted into the Gt(ROSA)26Sor locus on chromosome 6. The "MADM TG" cassette has an ATG start codon, a beta-globin intronic sequence (containing one frt and several lox sites), and a C-terminal portion of mut4EGFP. Mice homozygous for the R26^TG allele are viable and fertile, with no gross behavioral or observable abnormalities. Homozygous mice exhibit no fluorescent protein expression in absence of its reciprocal mutation (even if Cre or FLP recombinase is present).

The new TG cassette in R26^TG mice is compatible with any "GX cassette" (GFP^N-terminus-intron-X^ATG-less) where X^ATG-less is any gene without the start codon.

To combine MADM with the Tet-Off binary expression system, R26^TG mice must be crossed to mice harboring the R26^G-tTA2 allele (Stock No. 017909) to generate Cre or FLP recombinase-induced fluorescent labeling/tTA2-expression. A detailed description and figure of this MADM-Tet principle is available here.

For "new MADM-6" (new mosaic analysis with double markers on chromosome 6), R26^TG mice must be crossed to mice harboring a reciprocal mutation at the same locus (R26^GT mice; Stock No. 017912) to allow Cre or FLP recombinase-induced fluorescent labeling of cells. A detailed description and figure of this MADM-6 principle is available here.

For the MADM-6 design, when recombined to have the complete mut4EGFP protein, mut4EGFP fluorescence is visible without the need for immunostaining. The donating investigator also reports the "new MADM-6" design has several advantages compared to the original MADM-6 mice. Specifically, tdTomato-3Myc fluorescence is visible without the need for immunostaining. This allows direct fluorescent visualization of both GFP and tdTomato in live animals/cells: permitting genotypes of distinctly labeled cells in mosaic animals to be unequivocally determined prior to fixation and/or immunostaining. Also, "new MADM-6" contains both lox sites and frt sites; allowing the induction of MADM-labeling by either Cre or FLP recombinase introduction in cell phase G0 or G1. The donating investigator did not specifically test FLP recombinase-mediated interchromosomal recombination efficiency. Another advantage to the "new MADM-6" design is that the beta-actin intron contains a frt-flanked region of alternate lox sites. These lox versions, lox5171 and lox2272, are compatible only with a lox sequence identical to self; they do not recombine with each other or with loxP sites. These were included in an attempt to further increase recombination efficiency. However, the donating investigator has not yet performed specific comparisons to date (April 2012) to test if this new configuration results in increased recombination efficiency compared to the original MADM-6.

Development

A targeting vector was created with a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "MADM TG" cassette. The "MADM TG" cassette used here (ATG-intron-GFP^C-terminus) was designed with an ATG start codon, a beta-globin intronic sequence (containing a frt-flanked region of two lox site variants [lox2272 and lox5171] and then a loxP-flanked neomycin resistance gene), the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP second exon; nucleotides 275-735 including stop codon), and an SV40 T-antigen poly(A) signal. This entire construct was subcloned into the pROSA26-PA targeting vector to generate the final targeting construct pROSA26-TG. The pROSA26-TG 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 into recipient blastocysts. Chimeric progeny were bred to either CD1 or C57BL/6J mice to generate the colony. The resulting R26^TG mice were bred to animals of mixed genetic background (mixed CD1, 129, C57BL/6 and/or FVB/N) for several generations prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish The Jackson Laboratory Repository colony.

Allele

Symbol: Gt(ROSA)26Sor^{tm7(ACTB-EGFP*)Luo}
Name: targeted mutation 7, Liqun Luo
MGI accession ID: MGI:5314253
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
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⁺
Site of expression: These mice contain somatic cells of different genotypes which have mosaic EGFP expression in daughter cells when using the "new MADM-6" system.
Molecular note: A targeting vector was created with a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "MADM TG" cassette. The "MADM TG" cassette used here (ATG-intron-GFPC-terminus) was designed with an ATG start codon, a beta-globin intronic sequence (containing a frt-flanked region of two lox site variants [lox2272 and lox5171] and then a loxP-flanked neomycin resistance gene), the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP second exon; nucleotides 275-735 including stop codon), and an SV40 T-antigen poly(A) signal. This entire construct was subcloned into the pROSA26-PA targeting vector to generate the final targeting construct pROSA26-TG. The pROSA26-TG construct was inserted into the Gt(ROSA)26Sor locus.