STOCK Igs5^{tm2.1(ACTB-EGFP,-tdTomato)Luo}/J

Stock number: 021457
Product name: MADM-7^GT (Hipp7^GT)
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

The MADM-7^GT (Igs5^GT) allele has the CMV enhancer/chicken beta-actin core promoter, the N-terminal portion of mut4-EGFP, a beta-globin intronic sequence (containing frt and lox sites), and the MYC-tagged C-terminal portion of tdTomato all inserted into the Igs5 locus on chromosome 7 (~2.13 cM; ~0.7 kbp downstream of exon 5 of the Rps9 gene). These MADM-7^GT mutants are designed for MADM (mosaic analysis with double markers), and must be crossed to MADM-7^TG mice harboring a reciprocal mutation at the same locus (Stock No. 021458). This MADM system 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, mitosis, and imprinting.

Detailed description

Mice homozygous for the MADM-7^GT (Igs5^GT) allele are viable and fertile with no reported abnormalities. The MADM-7^GT allele has the CMV enhancer/chicken beta-actin core promoter, the N-terminal portion of mut4-EGFP, a beta-globin intronic sequence (containing a frt-flanked region of two lox site variants [lox2272 and lox5171] and then a single loxP site), and the MYC-tagged C-terminal portion of tdTomato all inserted into the Igs5 locus on chromosome 7 (~2.13 cM; ~0.7 kbp downstream of exon 5 of the Rps9 gene). These MADM-7^GT mutants are designed for MADM (mosaic analysis with double markers), and must be crossed to MADM-7^TG mice harboring a reciprocal mutation at the same locus (Stock No. 021458). 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 Hipp7 locus on chromosome 7, 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-7 system are listed below. Because of its placement ~3.6 Mbp from the centromere, MADM-7 allows almost all of the genes on chromosome 7 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-7 design has several advantages compared to the original MADM(-6) mice. Specifically, MADM-7 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-7 contains both loxP and frt sites; allowing the induction of MADM-labeling by either Cre recombinase or FLP recombinase.

The same donating investigator has several mice with MADM applications on different chromosomes:
On chromosome 6, the Gt(ROSA)26Sor knockin mutations include MADM-6^GR (Stock Nos. 006041 / 006075), MADM-6^RG (Stock Nos. 006067 / 006080), MADM-6^GG (Stock Nos. 006053 / 006071), R26^GT (Stock No. 017912), R26^TG (Stock No. 017921), R26^TT (Stock No. 017922), and R26^G-tTA2 (Stock No. 017909).
On chromosome 7, the centromeric insertions are MADM-7^GT (Igs5^GT; Stock No. 021457) and MADM-7^TG (Igs5^TG; Stock No. 021458).
On chromosome 10, the centromeric insertions are MADM-10^GT (Igs3^GT; Stock No. 017923) and MADM-10^TG (Igs3^TG; Stock No. 017932).
On chromosome 11, the centromeric insertions are MADM-11^GT (Hipp11^GT; Stock No. 013749) and MADM-11^TG (Hipp11^TG; Stock No. 013751).
On chromosome 12, the centromeric insertions are MADM-12^GT (Igs6^GT; Stock No. 021460) and MADM-12^TG (Igs6^TG; Stock No. 021461).

Development

A targeting vector was created with a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "GT MADM" cassette. The "GT MADM" cassette used here was designed with the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; aa 1-273), 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 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 Igs5 locus on chromosome 7 (~2.13 cM; ~0.7 kbp downstream of exon 5 of the Rps9 gene) 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 males were bred to CD1 females to establish the MADM-7^GT colony. The donating investigator reports that MADM-7^GT mice were previously bred to Hprt-Cre mice (on an undisclosed genetic background; see Stock No. 004302), and the floxed neomycin resistance gene was removed. The donating investigator also reports that the MADM-7^GT mice no longer have the Cre recombinase gene. MADM-7^GT mice on a mixed CD1;129;C57BL/6 genetic background (white coat color) were sent to The Jackson Laboratory Repository in 2013. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). The MADM-7^GT mice were then bred together, to wildtype mice from the colony, or to C57BL/6J mice.

Allele

Symbol: Igs5^{tm2.1(ACTB-EGFP,-tdTomato)Luo}
Name: targeted mutation 2.1, Liqun Luo
MGI accession ID: MGI:5470395
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Igs5
Marker name: intergenic site 5
Chromosome: 7
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: EGFP and tdTomato will be expressed in cells/tissues where promoters driving Cre recombinase and Flp recombinase are expressed.
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 Iis5 locus locate at about 0.7 kb downstream of exon 5 of the Rps9 gene at 2.13cM. Mice were bred to Hprt-Cre mice (on an undisclosed genetic background) and the floxed neomycin resistance gene was removed.