STOCK Igs2^{tm1(ACTB-EGFP,-tdTomato)Zng}/Igs2^{tm2(ACTB-tdTomato,-EGFP)Zng}/J

Stock number: 030578
Product name: GT11ML/TG11ML (MADM-ML-11^GT/TG) trans-heterozygous
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

GT11ML/TG11ML (MADM-ML-11^GT/TG compound heterozygous mice harbor reciprocal mutations on homologous chromosomes ("trans-heterozygous"), and are designed for MADM (mosaic analysis with double markers). Exposure to Cre- or FLP-recombinase induces fluorescent protein expression.
Compared to the single loxP approach of the original MADM designs, the multiple self-recognizing lox variant sites present in the MADM-ML 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). This MADM-ML 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

The GT11ML/TG11ML (MADM-ML-11^GT/TG) compound heterozygous mice harbor reciprocal mutations on homologous chromosomes ("trans-heterozygous"). Specifically, the GT11ML targeted mutation (MADM-ML-11^GT ; Igs2^{tm1(ACTB-EGFP,-tdTomato)Zng}) is on one homologous chromosome, and the TG11ML targeted mutation (MADM-ML-11^TG ; Igs2^{tm2(ACTB-tdTomato,-EGFP)Zng}) is on the other homologous chromosome.
Each respective allele has the CMV enhancer/chicken beta-actin core promoter upstream of reciprocal portions of mut4-EGFP and MYC-tagged tdTomato separated by a beta-globin intronic sequence (containing frt-lox5171-lox2272-frt-loxP-Neo-loxP) - all inserted into the Igs2 locus (Hipp11 or MADM-11; an intergenic region near the centromere of chromosome 11.
GT11ML/TG11ML mice are viable and fertile with no gross behavioral or observable abnormalities. These GT11ML/TG11ML trans-heterozygotes are designed for MADM (mosaic analysis with double markers), and must 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 Igs2 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 studies of cell differentiation, mitosis, and imprinting.

Other important features of the MADM-ML-11 system are listed below:
Compared to the single loxP approach of the original MADM-11 designs (MADM-11^GT [Stock No. 013749] and MADM-11^TG [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). Because of its placement ~3 kbp from the centromere, MADM-ML-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. Similar to the MADM-11 design, MADM-ML-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-ML-11 contains multiple lox sites and two frt sites; allowing the induction of MADM-labeling by either Cre recombinase or FLP recombinase.

Development

The GT11ML/TG11ML (MADM-ML-11^GT/TG) compound heterozygous mice (Stock No. 030578) harbor reciprocal mutations on homologous chromosomes ("trans-heterozygous"). They were created in 2017 by breeding GT11ML mice (Stock No. 022976) with TG11ML mice (Stock No. 022977). Each allele is described below.

The GT11ML targeted mutation (MADM-ML-11^GT ; Igs2^{tm1(ACTB-EGFP,-tdTomato)Zng}) was designed by Dr. Hui Zong (University of Virginia). To create the "multiple lox" MADM-ML-11^GT targeting vector, the single-loxP MADM-11^GT targeting vector (created by Dr. Hui Zong while a member of Dr. Liqun Luo's laboratory at Stanford University/HHMI) was obtained and modified to have multiple lox sites as described below. The "multiple lox" MADM-ML-11^GT targeting vector had a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "GT MADM-ML" cassette. The "GT MADM-ML" cassette used here was designed with the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; nucleotide 1-273), a beta-globin intronic sequence (containing an frt-lox5171-lox2272-frt-loxP-Neo-loxP), the C-terminal portion of a red fluorescent protein (tdTomato; nucleotide 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-ML" construct was inserted into the Igs2 locus (Hipp11 or MADM-11; an intergenic region on mouse chromosome 11 [cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci]). Mice harboring the GT11ML (MADM-ML-11^GT) allele on a mixed C57BL/6;CD1;129 genetic background from Stock No. 022976 were used for creating the GT11ML/TG11ML double mutant animals.

The TG11ML targeted mutation (MADM-ML-11^TG ; Igs2^{tm2(ACTB-tdTomato,-EGFP)Zng}) was designed by Dr. Hui Zong (University of Virginia). To create the "multiple lox" MADM-ML-11^TG targeting vector, the single-loxP MADM-11^TG targeting vector (created by Dr. Hui Zong while a member of Dr. Liqun Luo's laboratory at Stanford University/HHMI) was obtained and modified to have multiple lox sites as described below. The "multiple lox" MADM-ML-11^TG targeting vector had a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "TG MADM-ML" cassette. The "TG MADM-ML" cassette used here was designed with the N-terminal portion of a red fluorescent protein (tdTomato; nucleotide 1-3), a beta-globin intronic sequence (containing an frt-lox5171-lox2272-frt-loxP-Neo-loxP), the C-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; nucleotide 274-724), and an SV40 T-antigen poly(A) signal. This entire "TG MADM-ML" construct was inserted into the Igs2 locus. Mice harboring the TG11ML (MADM-ML-11^TG) allele on a mixed C57BL/6;CD1;129 genetic background from Stock No. 022977 were used for creating the GT11ML/TG11ML double mutant animals.

Allele

Symbol: Igs2^{tm1(ACTB-EGFP,-tdTomato)Zng}
Name: targeted mutation 1, Hui Zong
MGI accession ID: MGI:5520969
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: GT11ML; MADM-ML
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
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: EGFP and tdTomato will be expressed in cells/tissues where promoters driving Cre recombinase and Flp recombinase are expressed.
Molecular note: The "multiple lox" MADM-ML-11 targeting vector has a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "GT MADM-ML" cassette. The "GT MADM-ML" cassette is designed with the N-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; nucleotide 1-273), a beta-globin intronic sequence (containing an frt-lox5171-lox2272-frt-loxP-Neo-loxP), the C-terminal portion of a red fluorescent protein (tdTomato; nucleotide 4-1544) tagged with three copies of the Myc epitope at its C-terminus, and an SV40 T-antigen poly(A) signal.

Allele

Symbol: Igs2^{tm2(ACTB-tdTomato,-EGFP)Zng}
Name: targeted mutation 2, Hui Zong
MGI accession ID: MGI:5520975
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: MADM-ML; TG11ML
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
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: EGFP and tdTomato will be expressed in cells/tissues where promoters driving Cre recombinase and Flp recombinase are expressed.
Molecular note: The "multiple lox" MADM-ML-11 targeting vector has a CMV enhancer/chicken beta-actin core promoter (pCA) upstream of the "TG MADM-ML" cassette. The "TG MADM-ML" cassette is designed with the N-terminal portion of a red fluorescent protein (tdTomato; nucleotide 1-3), a beta-globin intronic sequence (containing an frt-lox5171-lox2272-frt-loxP-Neo-loxP), the C-terminal portion of a mutant enhanced green fluorescent protein (mut4-EGFP; nucleotide 274-724), and an SV40 T-antigen poly(A) signal.