Figure 1.
The MADM principle and design of new MADM cassettes.
A) MADM relies on two reciprocally chimeric marker genes (for example, GR and RG, see part B below for cassette description) that have been knocked into the same locus on homologous chromosomes. Recombination in the G2 phase of the cell cycle regenerates the functional marker genes on a pair of chromatids. X-segregation of chromatids (the recombinant chromatids segregate to different cells) generates a red and a green cell. Z-segregation of chromatids (the recombinant chromatids congregate to the same cell) generates a double-labeled (yellow) cell and an unlabeled cell. If a mutation (asterisk) is present distally to the GR cassette, the green cells will be homozygous for the mutation. This orientation of the cassettes corresponds to MADM in the Rosa26 locus. If the cassettes are in the opposite orientation with respect to the centromere, the genotypes for green and red cells will be inverted (for example in MADM-11). If mitotic recombination occurs in G0 or G1, a double-labeled cell is produced without altering the genotype of the cell. B) The “old” MADM cassettes contained two genes encoding fluorescent proteins (dsRed2 and GFP) split roughly in the middle. The “new” cassettes use the same GFP split, but split the second gene (for example, tdTomato) into ATG and GeneATG-less. That way, the ATG-GC-terminus (for simplicity, TG) becomes a universal cassette that can be paired with any G-GeneATG-less cassette. The single white triangle represents a single loxP site, a combination of loxP sites or the loxP-flanked (floxed) neomycin resistance gene (see Figure S1 for detailed description of MADM cassettes).
Figure 2.
Random integration-based approach to expand MADM to other mouse chromosomes.
A and B) Schematic representations of MADM precursor (pMADM) constructs. A) pMADMα contains the CA promoter, FRT-flanked MADM GT and TG cassettes and a single polyadenylation signal (pA). The cassette containing the floxed neomycin phosphotransferase gene (loxP-pPGK-Neo-pA-loxP) is placed in the introns of both cassettes. pMADMα can be converted into either GT or TG via partial Flp-mediated recombination in ES cells. B) pMADMβ construct contains the CA promoter driving the βgeo gene (a lacZ and neomycin-phosphotransferase fusion) flanked by FRT5 and FRT. pMADMβ can be converted into any transgene, including a GT or TG cassette via Flp- and FRT5/FRT-mediated cassette exchange in ES cells. These MADM cassettes contained a hygromicin resistance gene (H) that was removed by φC31 integrase-mediated recombination (see Figure S1) before performing the experiments shown in D. C) Distribution of pMADM transgene intergenic integration sites in the mouse genome. Each centromere is represented by a blue circle, and mapped insertion sites are indicated by triangles (Mb, mega base pair). The pMADMα insertion site used to establish MADM-10 (Figure 2D) is represented by the blue triangle located close to the centromere of Chr. 10. All the other triangles represent the insertion sites of pMADMβ transgenes based on the 5′ genomic sequence amplified by Splinkerette PCR. Insertion sites that were mapped close to centromeres, and were independently confirmed by both 5′ and 3′ genomic PCR, are represented by red triangles. The insertion located ∼39 Mb from the centromere of Chr. 1 (indicated by an asterisk) was used to establish MADM-1. D) Representative epifluorescence images of tissue sections with genotypes indicated on top and tissue identity on the bottom. The sections were unstained or stained only with DAPI to label nuclei (blue). The creation of fluorescent cells was Cre-dependent. Scale bars, upper row of images: 100 µm, lower row: 50 µm.
Figure 3.
Targeted knock-in approach to create new Rosa26 MADM with GT and TG cassettes.
A) Schematic representation of new alleles: R26GT and R26TG. B), C) and D) Representative confocal images from tissues indicated on the bottom and genotypes indicated on top. Expected labeling was observed only when Cre was present (compare B with C and D). Bright cellular labeling observed in C and D originates from native tdT and GFP fluorescence (no additional immunostaining was performed). Some sections were stained with DAPI to label nuclei (blue). Scale bars, 50 µm.
Figure 4.
Test for global, biallelic expression from the newly modified MADM loci by creation of GG/TT transheterozygotes.
A) Mating scheme outlines the creation of GG and TT alleles via Cre-mediated meiotic recombination. The two new lines for each locus were crossed to each other to generate the transheterozygous GG/TT animals. B) Representative confocal images of unstained tissue sections obtained from animals with genotypes represented above. Cells or groups of cells, in which the expression of one marker is markedly higher than the expression of the other, are indicated by asterisks. Scale bars, 100 µm.
Figure 5.
Cells with translocations and aneuploidy generated and labeled by MADM in vivo.
A) Schematic representation of cellular genotypes generated by interchromosomal recombination between non-homologous Chr. 6 and Chr. 10. In both chromosomes, the MADM cassettes are oriented in the telomere-to-centromere fashion. Each double-labeled cell contains the same reciprocal translocation, resulting in no net loss or gain of DNA. Single-labeled (green and red) cells exhibit abnormal copy numbers for parts of the chromosomes distal to the loxP sites: red cells are monosomic for the Chr. 6 portion and trisomic for the Chr. 10 portion; green cells have the reciprocal trisomy/monosomy. B) Representative confocal images of tissue sections obtained from R26GT/+;M10TG/+;HprtCre/Y mice. The sections were unstained or stained only with DAPI to label nuclei (blue, in the olfactory epithelium panel). The insets within the olfactory epithelium panel show examples of twin-spot labeling where red and green cells are located in close proximity. Due to the overall low frequency of labeling, each twin-spot labeling most likely originated from a single mitotic recombination event. Scale bars, panels: 100 µm, insets: 25 µm. C) Schematic representation of cellular genotypes generated by interchromosomal recombination between non-homologous Chr. 10 and Chr. 11. The MADM cassettes are oriented differently in the two chromosomes with respect to the corresponding centromeres. Each double-labeled cell contains the reciprocal translocation, resulting in one acentric and one dicentric chromosome. Single-labeled cells contain a dicentric or an acentric chromosome, and also exhibit abnormal copy numbers; the red cells are trisomic for Chr. 11 portion distal to loxP and monosomic for Chr. 10 portion proximal to loxP; the green cells are monosomic for Chr. 11 portion distal to loxP and trisomic for Chr. 10 portion proximal to loxP. D) Representative confocal images of unstained tissue sections obtained from M10TG/+;H11GT/+;HprtCre/Y mice. Scale bars, 100 µm.
Figure 6.
MADM-Tet combines MADM with a binary expression system.
A) Schematic representation of MADM-Tet starting with the following genotype: R26TG/G-tTA2;Nestin-Cre+/−;TRE-KZ+/−. Although all cells contain the Nestin-Cre and TRE-KZ transgenes, for simplicity they are displayed within the cells only when they are active. B) Confocal images of tissue sections stained with antibodies against GFP (green) and lacZ (red) from mice of the genotype indicated above. Note that the two markers exhibit different subcellular distribution: GFP labels whole cells including nuclei, whereas tau-lacZ is absent from the nuclei and labels the processes more strongly (an example of a red-only cell body is indicated by an arrowhead). Scale bars, left and middle panels: 50 µm, right panel: 25 µm.