Phenotypic and molecular analysis of the compound <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mouse model.

<p>(A) Schematic illustration of the chr. 18 LOH event in intestinal tumors from <i>in cis Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice leading to loss of both <i>Smad4</i> and <i>Apc</i> wild-type alleles. (B) Comparative phenotypic analysis of the intestinal and mammary tumor predisposition among <i>Apc</i>^+/1572T, <i>Smad4</i>^+/Sad, and <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice. <i>Notes</i>: (1) The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count. (2) The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible. The asterisks indicate that the apparent absence of intestinal tumor in <i>Smad4</i>^+/Sad control animals is not in contradiction with what previously published. These mice were sacrificed at time points matched with the ages at which compound <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice had to be sacrificed due to the high GI and mammary tumor burden (♀: 90.4 days +/−28.4; ♂: 118.5 days +/−26.2). However, in <i>Smad4</i>^+/Sad animals the majority of the tumors appear at 9 months of age <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Alberici1" target="_blank">[15]</a>. (C) H&E staining of intestinal tumor sections from <i>Apc</i>^+/1572T (top), <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad (middle), and <i>Smad4</i>^+/Sad (bottom) mice. (D) Smad4 IHC analysis of two intestinal adenomas from <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad mice showing loss of Smad4 expression. LOH was observed in 100% of the polyps (n = 15) analyzed. PCR–based LOH analysis of the same cohort of <i>Apc</i>^+/1572T/<i>Smad4</i>^+/Sad polyps revealed loss of wild-type <i>Apc</i> allele in 87% of the cases (13/15; data not shown).</p

Overview of the phenotypic comparisons between <i>Apc</i>^+/1572T and other <i>Apc</i>-mutant mouse models.

<p>Notes: Incidence is given as percentage of affected animals.</p>1,3<p>Animals were sacrificed when signs of discomfort were apparent and/or when tumor size reached 2 cm.</p>2<p>The incidence of GI tumors was calculated after exclusion of the pyloric lesions as these present in clusters often difficult to count.</p>3<p>The multiplicity of GI tumors was calculated based on all animals with the exception of those where the high tumor burden made the count not feasible.</p>*<p>This specific animal was found to carry a single tumor at 21 months of age, likely to represent a sporadic case. Background (B) of the different strains analyzed: F1: C57Bl6/J x 129Ola; Ola: inbred 129Ola; B6: inbred C57Bl6/J. n.d. not determined.</p

Differentiation.

<p>HE staining of mammary tumors (A–B) and pulmonary metastases (C) from <i>Apc</i>^+/1572T mice shows typical mammary glandular architecture and squamous differentiation. (D–F) Luminal epithelial differentiation as shown by cytokeratin 8 (Ck8) IHC staining. (G–I) Myoepithelial differentiation revealed by IHC staining with the Sma antibody. (J–L) IHC analysis with antibodies directed against cytokeratin 14 (Ck14) confirm the presence of squamous differentiation (hair follicle and skin cellular types). (M–O) β-catenin IHC analysis shows heterogeneous subcellular localization and intracellular accumulation with fewer cells characterized by positive nuclear staining. The results shown in this figure were confirmed in 12 independent primary tumors.</p

LOH analysis of <i>Apc</i>^+/1572T mammary adenocarcinomas.

<p>(A) PCR–based LOH analysis of tumour DNA samples amplified in the presence of radioactive nucleotides as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Smits3" target="_blank">[35]</a>. Samples were scored as having lost the wild type allele when the ratio between the intensity of the two alleles was ≤0.6. <i>Apc</i>^+/1572T mammary tumor samples (T); <i>Apc</i>^+/+ DNA control (C₁); <i>Apc</i>^+/1572T DNA control (C₂). Out of the 27 samples (collected from 23 tumours) analysed, only two (lanes marked by an asterisks) show a ratio higher than 0.6 and were accordingly scored as not having allelic imbalance. (B) Western analysis of tumour-derived total protein lysates confirms the somatic loss of full length Apc. Tumour samples (T); wild type control (C₃); <i>Apc</i>^+/1572T control from tail sample (C₄).</p

Phenotypic characterization of <i>Apc</i>^+/1572T mice: mammary adenocarcinomas are composed by mixed differentiation lineages with heterogeneous patterns of β-catenin intracellular accumulation and subcellular localization.

<p>Survival curves of (A) female and (B) male <i>Apc</i>^+/1572T mice, respectively. The black, green and red lines are representative of mice in the 129Ola, F1 B6x129Ola, and B6 respectively. Please note that in these graphs, age of death represents the moment at which, due to the presence of signs of discomfort or because the tumor size exceeded 2 cm³, mice had to be euthanized according to institutional and national regulations. (C) Macroscopic image of the appearance of the mammary adenocarcinomas characteristic of the <i>Apc</i>1572T model. (D) Examples of global digital microscopy scans of two mammary adenocarcinomas from <i>Apc</i>^+/1572T mice illustrative of the multi-lineage nature of these lesions.</p

Teratoma formation assays indicate an intermediate differentiation defect in <i>Apc</i>^1572T/1572T ES cells.

<p>(A) HE analysis of normal mammary gland with luminal cells surrounded by a basal layer of myoepithelial cells. (B) IF analysis of normal mammary glands for Ck8 (luminal cells, green) and Sma (myoepithelial, red). (C) HE staining of <i>Apc</i>^1572T/1572T teratoma showing the typical mammary gland architecture with lobular and ductal structures. (D) IF analysis of <i>Apc</i>^1572T/1572T teratomas for luminal and myoepithelial cell types. The frequency of these structures in teratomas derived from <i>Apc</i>^1572T/1572T ES cells is largely increased when compared with (F) teratomas derived from <i>Apc</i>^+/+ ES cells. (E) Summary of the results of the teratoma differentiation assays of <i>Apc</i>-mutant ES cells. Antibodies employed to evaluate differentiation are: Glial Fibrillary Acidic Protein (GFAP) for glial cells; 2H3 for neurofilaments; SV2 for synaptic vesicles; A4.1025 for adult myosin. Mammary gland structures were primarily identified by HE and then confirmed by IF as shown in panels a–d; also in the case of cartilage and epithelial structures HE stained section were employed. n.d. not determined. Differentiation levels were scored as: (−) not present; (−*) vestigial presence; (+) present; (++) highly abundant. The shaded areas indicate groups of teratomas for which the corresponding antibody staining was negative.</p

Biochemical characterization of the targeted <i>Apc</i>1572T allele.

<p>(A) Schematic representation of the APC tumor suppressor protein, its functional domains, and the truncated proteins resulting from the <i>Apc</i>1572T, <i>Apc</i>1638N, and <i>Apc</i>1638T targeted alleles. Only residual amounts (2%) of the truncated Apc1638N protein are encoded by the targeted allele, as shown by immuno-precipitation analysis of <i>Apc</i>^1638N/1638N ES lines <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Kielman1" target="_blank">[5]</a>. (B) β-catenin/TCF reporter assay (TOP-FLASH) analysis of <i>Apc</i>^+/+ (1) and <i>Apc</i>-mutant ES cell lines: Apc^1638T/1638T (2); Apc^1572T/1572T (3); Apc^1638N/1638N (4). Each bar represents the average measurement of the luciferase units from triplicate assays. For each cell line, 3 independent experiments were performed with the TOP (filled bars) and FOP (empty bars) reporter constructs. The bold figures represent the average TOP/FOP ratio of all independent experiments. Depicted error bars correspond to standard deviation. In brief, ES cells were plated on dishes coated with MEFs and subsequently transfected by lipofection with either the TOP-FLASH or FOP-FLASH reporter constructs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000547#pgen.1000547-Korinek1" target="_blank">[10]</a> together with the Renilla luciferase vector for normalization purposes. (C) Immuno-precipitation (IP) analysis of Apc-bound β-catenin in <i>Ap</i>c-mutant ES cell lines. For comparative purposes, immuno-precipitates obtained from equal amounts of total cellular lysates were loaded.</p

Dystrophin expression pattern and morphological examination of del52hDMD/<i>mdx</i> mouse lines.

<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p

PCR analysis of targeted clones and confirmation of deletion exon 52 on RNA level.

<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p

Additional file 1: of The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation

Supplemental materials. (DOCX 2768 kb