Random integration-based approach to expand MADM to other mouse chromosomes.

<p><b>A and B</b>) Schematic representations of MADM precursor (<i>pMADM</i>) constructs. <b>A</b>) <i>pMADMα</i> contains the <i>CA</i> promoter, <i>FRT</i>-flanked MADM <i>GT</i> and <i>TG</i> cassettes and a single polyadenylation signal (<i>pA</i>). The cassette containing the floxed neomycin phosphotransferase gene (<i>loxP-pPGK-Neo-pA-loxP</i>) is placed in the introns of both cassettes. <i>pMADMα</i> can be converted into either <i>GT</i> or <i>TG</i> via partial Flp-mediated recombination in ES cells. <b>B</b>) <i>pMADMβ</i> construct contains the <i>CA</i> promoter driving the <i>βgeo</i> gene (a <i>lacZ</i> and neomycin-phosphotransferase fusion) flanked by <i>FRT5</i> and <i>FRT</i>. <i>pMADMβ</i> can be converted into any transgene, including a <i>GT</i> or <i>TG</i> cassette via Flp- and <i>FRT5/FRT</i>-mediated cassette exchange in ES cells. These MADM cassettes contained a hygromicin resistance gene (<i>H</i>) that was removed by φC31 integrase-mediated recombination (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033332#pone.0033332.s001" target="_blank">Figure S1</a></b>) before performing the experiments shown in D. <b>C</b>) Distribution of <i>pMADM</i> transgene <i>intergenic</i> 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 <i>pMADMα</i> insertion site used to establish <i>MADM-10</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033332#pone-0033332-g002" target="_blank">Figure 2D</a>) is represented by the blue triangle located close to the centromere of Chr. 10. All the other triangles represent the insertion sites of <i>pMADMβ</i> 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 <i>MADM-1</i>. <b>D</b>) 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.</p

Cells with translocations and aneuploidy generated and labeled by MADM <i>in vivo</i>.

<p><b>A</b>) 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 <i>loxP</i> 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>B</b>) Representative confocal images of tissue sections obtained from <i>R26^GT/+;M10^TG/+;Hprt^Cre/Y</i> 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. <b>C</b>) 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 <i>loxP</i> and monosomic for Chr. 10 portion proximal to <i>loxP</i>; the green cells are monosomic for Chr. 11 portion distal to <i>loxP</i> and trisomic for Chr. 10 portion proximal to <i>loxP</i>. <b>D</b>) Representative confocal images of unstained tissue sections obtained from <i>M10^TG/+;H11^GT/+;Hprt^Cre/Y</i> mice. Scale bars, 100 µm.</p

The MADM principle and design of new MADM cassettes.

<p><b>A</b>) MADM relies on two reciprocally chimeric marker genes (for example, <i>GR</i> and <i>RG</i>, 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 <i>GR</i> cassette, the green cells will be homozygous for the mutation. This orientation of the cassettes corresponds to MADM in the <i>Rosa26</i> 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 <i>MADM-11</i>). If mitotic recombination occurs in G0 or G1, a double-labeled cell is produced without altering the genotype of the cell. <b>B</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 <i>ATG</i> and <i>Gene^ATG-less</i>. That way, the <i>ATG-G^C-terminus</i> (for simplicity, <i>TG</i>) becomes a universal cassette that can be paired with any <i>G-Gene^ATG-less</i> cassette. The single white triangle represents a single <i>loxP</i> site, a combination of <i>loxP</i> sites or the <i>loxP</i>-flanked (floxed) neomycin resistance gene (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033332#pone.0033332.s001" target="_blank">Figure S1</a></b> for detailed description of MADM cassettes).</p

Test for global, biallelic expression from the newly modified MADM loci by creation of <i>GG/TT</i> transheterozygotes.

<p><b>A</b>) Mating scheme outlines the creation of <i>GG</i> and <i>TT</i> alleles via Cre-mediated meiotic recombination. The two new lines for each locus were crossed to each other to generate the transheterozygous <i>GG/TT</i> animals. <b>B</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.</p

Targeted knock-in approach to create new <i>Rosa26</i> MADM with <i>GT</i> and <i>TG</i> cassettes.

<p><b>A</b>) Schematic representation of new alleles: <i>R26^GT</i> and <i>R26^TG</i>. <b>B), C) and D)</b> 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>B</b> with <b>C</b> and <b>D</b>). Bright cellular labeling observed in <b>C</b> and <b>D</b> 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.</p

MADM-Tet combines MADM with a binary expression system.

<p><b>A</b>) Schematic representation of MADM-Tet starting with the following genotype: <i>R26^TG/G-tTA2;Nestin-Cre^+/−;TRE-KZ^+/−</i>. Although all cells contain the <i>Nestin-Cre</i> and <i>TRE-KZ</i> transgenes, for simplicity they are displayed within the cells only when they are active. <b>B</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.</p

Expression of the CaMKIIβ throughout the brain by AAV-PHP.eB.

Volume-rendered and single-plane images of the brain expressing H2B-mCherry under hSyn1 promoter by the AAV (mCherry, green) counterstained with RD2 (red). A volume-rendered image is shown in the center. Single-plane and magnified images are shown for cerebral cortex, thalamus, hippocampus, midbrain, cerebellum, striatum, and olfactory bulb. Scale bar in the center image, 3 mm; other scale bars, 100 μm. AAV, adeno-associated virus; CaMKIIβ, calmodulin-dependent protein kinase IIβ; hSyn1, human synapsin-1. (TIFF)</p

Robust sleep induction by CaMKIIβ T287D mutant.

(A) Expression levels of endogenous CaMKIIβ and AAV-mediated transduced CaMKIIβ in the brain. Camk2bFLAG/FLAG represents homo knock-in mice in which the FLAG tag was inserted into the endogenous Camk2b locus. PBS: PBS-administrated mice. Immunoblotting against FLAG-tagged protein indicates that AAV-mediated expression of CaMKIIβ is lower than the expression level of endogenous CaMKIIβ. (B) Calculated transduction efficiency plotted against sleep duration. Transduction efficiency is an estimation of the number of AAV vector genomes present per cell in a mouse brain. After the SSS measurements, we purified the AAV vector genomes from the mice brains and then quantified them with a WPRE-specific primer set and normalized to mouse genomes. (C) Sleep transition profiles of mice expressing CaMKIIβ T287-related mutants shown in Fig 1F. The shaded areas represent SEM. (D) Sleep parameters during light or dark period of mice expressing CaMKIIβ T287-related mutants shown in Fig 1F. Multiple comparison tests were performed between all individual groups in each phase. (E, F) Sleep/wake parameters of mice expressing S114-related CaMKIIβ mutants (C) and S109-related CaMKIIβ mutants (D), averaged over 6 days. The shaded areas represent SEM. Multiple comparison tests were performed between all individual groups and resulted in no significant differences. The underlying numerical data can be found in S1 Data, and uncropped or raw image files for S3A Fig are provided in S2 and S3 Data files. Error bars: SEM, *p p p (PDF)</p

Time-of-day analyses for sleep parameters of mice with perturbed CaMKII activity.

(A) Sleep transition profiles of mice expressing the CaMKIIβ del mutant under hSyn1 promoter shown in Fig 2B and 2C. The shaded areas represent SEM. (B) Sleep parameters of mice expressing the CaMKIIβ del mutants shown in Fig 2B and 2C during light or dark period. Multiple comparison tests were performed between all individual groups in each phase. (C) Sleep transition profiles of mice expressing AIP2 or RARA mutant under hSyn1 promoter shown in Fig 2E and 2F. The shaded areas represent SEM. PBS: PBS-injected mice (n = 6). (D) Sleep parameters of mice expressing AIP2 or RARA mutant shown in Fig 2E and 2F during light or dark period. Multiple comparison tests were performed between all individual groups in each phase. The underlying data can be found in S1 Data. Error bars: SEM, *p p p hSyn1, human synapsin-1; (PDF)</p