STOCK Igs3^{tm1.1(ACTB-EGFP*)Luo}/J

Stock number: 017932
Product name: Miya10^TG (M10^TG)
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

The Miya10^TG (M10^TG) transgene has the CMV enhancer/chicken beta-actin core promoter upstream of a frt-flanked "MADM TG" cassette, all inserted into an intergenic region on chromosome 10 (~21 Mbp; between the Ahi1 and Myb loci). These mutant mice are designed for MADM-10 (mosaic analysis with double markers on chromosome 10), and provide a tool to generate genetic mosaics in which an individual organism contains somatic cells of different genotypes. This allows Cre 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.

Detailed description

The Miya10^TG (M10^TG) transgene has the CMV enhancer/chicken beta-actin core promoter upstream of a frt-flanked "MADM TG" cassette, all inserted into an intergenic region on chromosome 10 (~21 Mbp; between the Ahi1 and Myb loci). The "MADM TG" cassette has an ATG start codon, a beta-globin intronic sequence, and the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP). Homozygous M10^TG mice 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 recombinase is present).

These mutant mice are designed for MADM-10 (mosaic analysis with double markers on chromosome 10), and must be crossed to mice harboring a reciprocal transgene at the same locus (M10^GT mice; Stock No. 017923) to allow Cre recombinase-induced fluorescent labeling of cells. A detailed description and figure of this MADM-10 principle is available here.

Other important features of the MADM-10 system are listed below. Because of its placement ~21kb from the centromere, MADM-10 allows genes in the distal ~108 Mbp of chromosome 10 to be subjected to MADM-based mosaic analyses. MADM-10 allows direct fluorescent visualization of both mut4EGFP and tdTomato-3Myc in live animals/cells: permitting genotypes of distinctly labeled cells in mosaic animals to be unequivocally determined prior to fixation and/or immunostaining. There are some limitations to MADM-10 as well. The labeling efficiency of MADM-10 is qualitatively lower than other MADM systems (such as MADM-6 and MADM-11). Moreover, the Miya10 locus is not bi-allelically expressed in some organs (e.g., liver), while it appears to be bi-allelically expressed in others (heart and cerebellum). As MADM-based mosaic analysis critically depends on bi-allelic expression of two cassettes in the cells of interest, the donating investigator reports that MADM-10 can be used in heart and the cerebellum, but bi-allelic marker expression should be tested before application of the MADM-10 system to other cell populations.

Development

The pMADMα transgene was designed with the CMV enhancer/chicken beta-actin core promoter (pCA) promoter, a frt site, the "MADM GT" cassette, and second frt site, the "MADM TG" cassette, a third frt site, and an SV40 T-antigen poly(A) signal. The "MADM GT" cassette used here has the N-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP first exon; nucleotides 1-274), a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and ATG-less tdTomato sequence (nucleotides 3-1548 including stop codon) tagged with three copies of the Myc epitope at its C-terminus (tdT3Myc^ATG-less). The "MADM TG" cassette used here has an ATG start codon, a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP second exon; nucleotides 275-735 including stop codon). The transgene was electroporated into (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. ES cells were screened for intact single-copy transgenes with integration in intergenic regions close to any centromere. ES cells were identified with an intact single-copy of the transgene inserted into an intergenic region at ~21 Mbp on chromosome 10 between the Ahi1 and Myb loci; this new insertion locus was named Miya10 (or M10). To convert pMADMα to the "MADM GT" or "MADM TG" genotype, ES cells were transfected with a FLP-expressing plasmid.

The resulting ES cells with recombination between the first and second frt sites (retaining the "MADM TG" cassette and deleting the "MADM GT" cassette) were microinjected into recipient blastocysts. Chimeric progeny were bred to either CD1 or C57BL/6J mice. The resulting Miya10^TG (M10^TG) mice were bred to mice with germline Cre recombinase expression to remove the floxed neo cassette. M10^TG mice were maintained on a mixed genetic background (mixed CD1, 129, C57BL/6 and/or FVB/N) for several generations (and the Cre recombinase gene removed) 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: Igs3^{tm1.1(ACTB-EGFP*)Luo}
Name: targeted mutation 1.1, Liqun Luo
MGI accession ID: MGI:5314259
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Igs3
Marker name: intergenic site 3
Chromosome: 10
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Cre recombination results in tdTomato and EGFP mosaic expression in cre-expressing tissues when using the MADM-10 system.
Molecular note: The pMADMalpha transgene was designed with the CMV enhancer/chicken beta-actin core promoter (pCA) promoter, a frt site, the MADM GT cassette, and second frt site, the MADM TG cassette, a third frt site, and an SV40 T-antigen poly(A) signal. The MADM GT cassette used here has the N-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP first exon; nucleotides 1-274), a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and ATG-less tdTomato sequence (nucleotides 3-1548 including stop codon) tagged with three copies of the Myc epitope at its C-terminus (tdT3MycATG-less). The MADM TG cassette used here has an ATG start codon, a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP second exon; nucleotides 275-735 including stop codon). The transgene was electroporated into (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. ES cells were screened for intact single-copy transgenes with integration in intergenic regions close to any centromere. ES cells were identified with an intact single-copy of the transgene inserted into an intergenic region at ~21 Mbp on chromosome 10 between the Ahi1 and Myb loci; this new insertion locus was named Miya10 (or M10). The transgene inserted into an intergenic region at ~21 Mbp on chromosome 10 between the Ahi1 and Myb loci; this new insertion locus was named Miya10 (or M10). ]Cre-mediated recombination removed the neo cassette.