Targeted <i>Rdh10</i> gene trap and knockout allele.

<p>(A) Schematic diagram of <i>Rdh10</i> knockout allele obtained as ES cells from KOMP consortium and derivative alleles. For <i>Rdh10^βgeo</i> allele, a gene-trap splice acceptor <i>βgeo</i> cassette is introduced between exon 1 and exon 2 of <i>Rdh10</i> and loxP sites are introduced surrounding exon 2. For the <i>Rdh10^flox</i> allele, the gene trap cassette is excised returning function, conditionally, to the <i>Rdh10</i> gene. For the <i>Rdh10^delta</i> allele, exon 2 is deleted by Cre excision of the DNA between the loxP sites. (B) <i>Rdh10</i> RNA in situ hybridization of E9.5 wild type embryo reveals expression of Rdh10 in specific tissues. <i>Rdh10</i> RNA staining pattern n>10 embryos. (C) Staining E9.5 <i>Rdh10^βgeo</i> embryofor β-galactosidase activity (arrow) reveals gene trap transcript expressed in pattern similar to <i>Rdh10</i> RNA expression. <i>Rdh10</i> β-galactosidase staining pattern n>10 embryos. ba, branchial arch; ht, heart; lb, limb bud; op, optic vesicle; psm, presomitic mesoderm; s, somite.</p

<i>Rdh10^trex</i> and new <i>Aldh1a2^gri</i> mutant phenotype comparison.

<p>Wild type (A,D), <i>Rdh10^trex/trex</i> mutant (B,E), and <i>Aldh1a2^gri/gri</i> mutant (C,F) embryos were collected at E9.5 (A–C) and E10.5 (D–F). Formalin-fixed embryos were stained with DAPI and imaged by confocal microscopy. For each embryo, a Z-stack of confocal slices was collapsed to form a “pseudo-SEM” image. (A–F) For each mutant phenotype at each indicated stage n>5 embryos. (G) Sequence of <i>Aldh1a2</i> gene at junction between Exon 4 and Intron 4–5 with <i>grimace</i> mutation single base change indicated beneath. Exon 4 genomic DNA sequence is indicated by yellow highlight, intron 4–5 sequence indicated by pink highlight. (H) Histogram representing relative levels of <i>Aldh1a2</i> mRNA from wild-type, heterozygous <i>Aldh1a2^gri/+</i> and homozygous <i>Aldh1a2^gri/gri</i> embryos, as assessed by quantitative RT-PCR, demonstrating loss of <i>Aldh1a2</i> transcript in homozygous mutants. fl, forelimb bud; h, heart; ov, otic vesicle; ppa, posterior pharyngeal arches.</p

Phenotype and characterization of transcript of <i>Rdh10^delta</i> allele.

<p>(A,C) Wild type and (B, D) homozygous <i>Rdh10^delta/delta</i> embryos were collected at E9.5 (A–B) and E10.5 (C–D. (E9.5 mutant embryos n = 34; rightward turn ¾ complete n = 33. E9.5 with leftward turn n = 1. E10.5 mutant embryos n = 5). Formalin-fixed embryos were stained with DAPI and imaged by confocal microscopy. For each embryo, a Z-stack of confocal slices was collapsed to form a “pseudo-SEM” image. (E) Schematic diagram of <i>Rdh10</i> spliced mRNA exon structure, along with coding sequence for <i>Rdh10</i> wild-type (full length) and <i>Rdh10^delta</i> mutant (truncated) as determined by direct sequencing of reverse transcribed <i>Rdh10</i> mRNA. Yellow boxes indicate spliced exons of <i>Rdh10</i> mRNA. Dark blue arrow represent coding sequence of spliced wild type <i>Rdh10</i> mRNA, blue boxes beneath arrow symbolize truncated coding sequence resulting from splicing of exon 1 directly to exon 3. Nucleotide and protein sequence indicated beneath schematic diagram shows 3′ end of exon 1 spliced directly to 5′ end of exon 3, generating a premature stop codon. Pink box indicates exon 1 derived sequence, green box represents exon 3 derived sequence. (F) Histogram of elative level of <i>Rdh10</i> mRNA as assessed by reverse transcription qPCR.</p

Distribution of RA signaling in <i>Rdh10</i> and <i>Aldh1a2</i> mutant embryos.

<p>Embryos carrying the RARE-lacZ reporter transgene were collected at E8.5, E9.5 or E10.5 and stained for β-galactosidase activity to visualize RA signaling. Blue staining indicates presence of RA. (A–B) <i>Aldh1a2^gri/gri</i> and littermates at E10.5. Arrow indicates small amount of RA signaling detected in neural tube of mutant embryos, n = 13. (C–F) <i>Rdh10^delta/delta</i> and littermates at E8.5, n = 5. (G–J) <i>Rdh10^delta/delta</i> and littermate embryo at E9.5, n = 6. Embryos shown in lateral (G–H) or dorsal (I–J) view.</p

<i>Rdh10</i> is up-regulated by reduction of RA.

<p>(A) Histogram representing relative levels of <i>Rdh10</i> mRNA from wild type, heterozygous <i>Aldh1a2^gri/+</i> and homozygous <i>Aldh1a2^gri/gri</i> embryos, as assessed by quantitative RT-PCR, demonstrating elevated levels of <i>Rdh10</i> transcript in homozygous <i>Aldh1a2^gri/gri</i> mutants. (B) RNA <i>in situ</i> hybridization for expression of <i>Rdh10</i> mRNA in E8.5 wild type and <i>Aldh1a2^gri/gri</i> mutant embryos, demonstrating that the increased <i>Rdh10</i> expression occurs in a pattern similar to the normal expression pattern for the gene (mutant embryos n = 5). (C) Model depicting that metabolic product RA feeds-back to reduce expression of membrane bound <i>Rdh10</i> in mouse embryos. Pink lines represent that RDH10 and RDH reaction occur in membrane-bound compartment. Cytosolic ADH enzymes do not contribute to any physiologically relevant production of RA in the embryo.</p

Phenotype of <i>Rdh10^βgeo</i> gene trap allele.

<p>(A) Wild type and (B) homozygous <i>Rdh10^βgeo/βgeo</i> embryos were collected at E9.5 (<i>Rdh10^βgeo/βgeo</i> n = 9). Wild type (C) and <i>Rdh10^βgeo/trex</i> compound heterozygous (D) embryos were collected at E9.75 (n = 6). Formalin-fixed embryos were stained with DAPI and imaged by confocal microscopy. For each embryo, a Z-stack of confocal slices was collapsed to form a “pseudo-SEM” image.</p

Gadgets and Experimental Setup in the Luquillo CZO. Presented at CZO All Hands meeting 9-21-2014 to 9-24-2014.

<p>Gadgets and Experimental Setup in the Luquillo CZO Presented at CZO All Hands meeting  9-21-2014 to 9-24-2014.</p

Ribosomal DNA copy number loss and sequence variation in cancer

<div><p>Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a <b>loss</b> of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a <i>Pten</i>^-/- mouse model for leukemia have <b>lower</b> rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.</p></div

Proliferation, rRNA production, and protein synthesis are robust in <i>Pten</i>^-/- HSCs.

<p>(A). To measure proliferation, HSCs were plated in 100 μl fresh medium, and cultured for 5 days. The viable cell number was measured based on total cell number and trypan blue staining. Data shown was derived from 12 clones derived from two age matched female mice of each genotype. (B). HSCs were pulse labeled with ³H-uridine for the time indicated. RNA was isolated with TriZol reagent. 1 μg of total RNA was counted in a Beckman LS 6500 multipurpose scintillation counter to determine new rRNA production. Three clones were labeled to derive the standard deviation from two pairs of mice of each genotype from the same litter. Significance was calculated using an unpaired t test, the asterisk indicates p<0.05. (C). To measure global protein synthesis, HSCs were pre-cultured in medium lacking methionine, and pre-treated with 10 μM MG-132, a proteasome inhibitor, for 1 hour. HSCs were incubated with 30 μCi of ³⁵S-methionine for 1 hour. Incorporation of ³⁵S-methionine into proteins was quantified in a liquid scintillation counter. Clones were derived from two mice of each genotype, with three replicates per genotype. A t test was performed for statistical significance.</p

SNV analysis of rDNA loci in the eight cancer genomes from tumor/normal pairs.

<p>The match between the test genome and the reference genome sequence was scored at each bp for all genomes. SNVs common to matched pairs were considered “shared” and not included in the analysis in A-C. (A). The plot depicts unique SNVs per kb in the repeat in normal and cancer genomes, with the project number indicated on the x axis, and the allele number on each bar. (B). All unique SNVs for all projects were summed together to depict the SNVs per kb for the different regions of the repeat. The total number of SNVs in each region is annotated on each bar. (C). The number of unique SNVs identified in the 28S region are plotted by position relative to the 45S repeat in GenBank. Hotspots of variation are apparent. (D). The number of alleles for each shared SNVs in each genome pair was used to calculate an average allele number per genome. The average for the normal genome was subtracted from the average calculated for the matched tumor genome to yield the allele difference, plotted by project with results of a t test indicated for each (phs000341, 0.0467, phs000409, 0.7223, phs000414, 0.3458, phs000447, 0.0011, phs000530, 0.8773, phs000579, 0.9782, phs000598, 0.0144, phs000699, 0.0641).</p