STOCK Igs2^{tm1(ACTB-EGFP,-tdTomato)Luo}/J

Stock number: 013749
Product name: MADM-11^GT
Strain synonyms: HIPP11^GT
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

These intergenic site 2 (commonly referred to as MADM-11^GT) targeted mutants are useful for generating fluorescent double markers in mice for studies to ascertain lineal relationships and pleiotropic gene function in multicellular organisms, and in studies of cell differentiation and mitosis.
Of note, an improved MADM design, called MADM-ML-11, is available: GT11ML (Stock No. 022976) and TG11ML (Stock No. 022977). Compared to the single loxP approach of the original MADM-11 design (Stock No. 013749 and Stock No. 013751), the multiple self-recognizing lox variant sites present in the MADM-ML-11 system result in significantly improved recombination efficiency (4-8 fold higher) with no negative impact on G2-X segregation percentage (G2-X segregation produces the desired fluorescent homozygous mutant daughter cells).

Detailed description

Homozygous MADM-11^GT mice are viable and fertile with no gross behavioral or observable abnormalities. The MADM-11^GT allele has the CMV enhancer/chicken beta-actin core promoter, the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP), a beta-globin intronic sequence containing an frt site and a loxP-flanked neomycin resistance gene, and the MYC-tagged C-terminal portion of a red fluorescent protein (tdTomato) all inserted into the Hipp11 locus on chromosome 11 (cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci). These MADM-11^GT mutants are designed for MADM (mosaic analysis with double markers), and must be crossed to MADM-11^TG mice harboring a reciprocal mutation at the same locus (see Stock No. 013751). The resulting GT/TG offspring have one copy of each reciprocal mutation on homologous chromosomes ("trans-heterozygous"), and must also be bred to harbor a cre- or FLP-recombinase to induce fluorescent protein expression. Prior to Cre- or FLP-recombination, trans-heterozygous mutant mice do not have colored cells: the chimeric genes do not produce functional proteins because their coding sequences are interrupted by the beta-globin intron in different reading frames. After DNA replication (G2 phase) in double mutant mice, introduction of Cre- or FLP-recombinase 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 subsequent chromatid segregation (X or Z) determines daughter cell phenotype: recombinant sister chromatids into the same daughter cell (a G2-Z event) leads to double reporter expression or no reporter expression, while independent segregation into separate daughter cells (a G2-X event) leads to expression of either EGFP or tdTomato-MYC. If an additional targeted mutation of interest is introduced distal to the MADM-11 locus on chromosome 11, only homozygous cells will be singly labeled following G2 cre or FLP introduction. The homozygous mutant and wildtype cells can then be distinguished by which single reporter they express. Most heterozygous cells will be unlabeled, but some heterozygous cells will be yellow (both markers expressed). Reporter protein tissue specificity, expression levels, and frequency of recombination are thus determined by the promoter controlling Cre- or FLP-recombinase 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.

Other important features of the MADM-11 system are listed below. Because of its placement ~3kb from the centromere, MADM-11 allows >99% of genes on chromosome 11 to be subjected to MADM-based mosaic analyses. Cre- or FLP-recombinase introduction in cell phase G0 or G1 results in double reporter expression. The donating investigator also reports the MADM-11 design has several advantages compared to the original MADM(-6) mice. Specifically, MADM-11 allows direct fluorescent visualization of both EGFP 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, MADM-11 contains both loxP sites and a frt site; allowing the induction of MADM-labeling by either Cre recombinase or FLP recombinase. In addition, the interchromosomal recombination rate in MADM-11 is markedly increased compared with the original MADM(-6) system; allowing greater temporal control of clone induction if using concomitantly with an inducible Cre recombinase (or Flp recombinase).

Development

The GT MADM targeting construct was designed with a CMV enhancer/chicken beta-actin core promoter (pCA), the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; aa 1-273), a beta-globin intronic sequence containing an frt site and loxP-flanked neomycin resistance gene, the C-terminal portion of a red fluorescent protein (tdTomato; aa 4-1544) tagged with three copies of the Myc epitope at its C-terminus, and an SV40 T-antigen poly(A) signal. This entire GT MADM construct was inserted into the Hipp11 locus on chromosome 11 (cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci) via electroporation of (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were microinjected into recipient blastocysts and chimeric progeny were bred with C57BL/6 mice to establish the MADM-11^GT colony. MADM-11^GT mice were bred with CD1 outbred mice for at least three generations. MADM-11^GT mice on a mixed CD1;C57BL/6;129 genetic background were sent 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 colony.

Allele

Symbol: Igs2^{tm1(ACTB-EGFP,-tdTomato)Luo}
Name: targeted mutation 1, Liqun Luo
MGI accession ID: MGI:4837736
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Igs2
Marker name: intergenic site 2
Chromosome: 11
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: Cre recombination results in EGFP and tdTomato mosaic expression in cre-expressing tissues when using the MADM-11 system.
Molecular note: The GT MADM targeting construct was designed with a CMV enhancer/chicken beta-actin core promoter (pCA), the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; aa 1-273), a beta-globin intronic sequence containing an frt site and loxP-flanked neomycin resistance gene, the C-terminal portion of a red fluorescent protein (tdTomato; aa 4-1544) tagged with three copies of the Myc epitope at its C-terminus, and an SV40 T-antigen poly(A) signal. This entire GT MADM construct was inserted into the "Hipp11" locus on chromosome 11 (cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci).