Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

No abstract

No abstract

It has been reported that neutrophil extracellular traps (NETs) play important roles in non-infectious diseases. In ischemic stroke, neutrophils infiltrate damaged brain tissue soon after injury and aggravate inflammation. Using a rat permanent MCAO model, we showed citrullinated histone H3 + (CitH3, a marker of NETosis) induction in neutrophils in leptomeninges and in peripheral blood soon after MCAO. Entry of CitH3 + cells occurred through leptomeninges after 6 h of MCAO and these cells were observed in cerebral cortex from 12 h and subsequently in striatum. It is interesting to note that CitH3 + induction began in circulating neutrophils before they migrated to brain parenchyma and they were detected as intact or lysed form. High mobility group box 1 (HMGB1), a danger associated molecular pattern (DAMP) molecule, was accumulated massively in serum after permanent MCAO and plays a critical role in CitH3 inductions in neutrophils in brain parenchyma and in peripheral blood. Both the all-thiol and disulfide types of HMGB1 induced CitH3 via their specific receptors, CXCR4 and TLR4, respectively. Importantly, HMGB1 not only induced NETosis but was included as a part of the extruded NETs, and contribute to NETosis-mediated neuronal death. Therefore, it would appear a vicious cycle exists between neuronal cell death and NETosis and HMGB1 mediates detrimental effects exerted by this cycle. When NETosis was suppressed by a PAD inhibitor in MCAO animals, delayed immune cell infiltrations were markedly suppressed and damages in blood vessels were significantly mitigated. The study shows NETosis with the involvement of HMGB1 as a mediator in a vicious cycle aggravates inflammation and subsequent damage in the ischemic brain. Electronic supplementary material The online version of this article (10.1186/s40478-019-0747-x) contains supplementary material, which is available to authorized users.

We isolated MED25, which associates with retinoic acid (RA)-bound retinoic acid receptor (RAR) through the C-terminal nuclear hormone receptor (NR) box/LxxLL motif, and increases RAR/RXR-mediated transcription. When tethered to a promoter, MED25 showed intrinsic transcriptional activity in its PTOV domain, which is likely accomplished by direct association with CBP. Reporter assays using dominant negatives of MED25 demonstrated the importance of the N-terminal Mediator-binding and C-terminal domains in CBP and RAR/RXR binding, which affect MED25 activity. Downregulation of MED25 specifically reduced RAR but not thyroid hormone receptor (TR) activity. Stimulation of RAR by MED25 was correlated with enhanced RA cytotoxicity in vivo. Chromatin immunoprecipitation (ChIP) assays revealed the RA-dependent recruitment of MED25 to the RARbeta2 promoter. Recruitment of CBP and TRAP220 was diminished by the overexpression of a MED25 NR box deletion mutant, and by treatment with MED25 siRNA. Time-course ChIP assays indicated that CBP, together with RAR and MED25, is recruited early, whereas TRAP220 is recruited later to the promoter. Our data suggest that MED25, in cooperation with CBP and Mediators through its distinct domains, imposes a selective advantage on RAR/RXR activation.

clones were isolated from human and mouse genomic libraries. The SHP gene was composed of two exons interrupted by a single intron spanning approximately 1.8 kilobases in human and 1.2 kilobases in mouse. Genomic Southern blot analysis and fluorescence in situ hybridization of human metaphase chromosomes indicated that the SHP gene is located at the human chromosome 1p36.1 subband. The 5-flanking regions of human and mouse SHP genes were highly conserved, showing 77% homology in the region of approximately 600 nucleotides upstream from the transcription start site. Primer extension analysis was carried out to determine the transcription start site of human SHP to 32 nucleotides downstream of a potential TATA box. The human SHP gene was specifically expressed in fetal liver, fetal adrenal gland, adult spleen, and adult small intestine. As expected from this expression pattern, the activity of the mouse SHP promoter measured by transient transfection was significantly higher in the adrenal-derived Y1 cells than HeLa cells.The nuclear receptor superfamily is a group of transcription factors regulated by small hydrophobic hormones such as retinoic acid, thyroid hormone, and steroids and also includes a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (for reviews see Refs. 1, 2). The nuclear receptors directly regulate transcription by binding to specific DNA sequences named hormone response elements, generally located in promoters of target genes. The nuclear hormone receptors share a common domain structure. The central DNA binding domain (DBD) 1 includes two zinc binding modules, which consist of a series of invariant cysteine residues. A conserved helical region termed the P box within the DBD (3) makes base-specific contacts and serves as one of the main criteria used for classification of the nuclear receptor superfamily. The C-terminal ligand binding domain (LBD) binds to the cognate ligands. This domain also contains dimerization and transcriptional activation functions. A less well conserved hinge domain that separates DBD and the ligand binding domain has been thought to serve merely as a flexible linker. However, recent results demonstrate that it is also involved with transcriptional repression, at least for a subset of receptors (4). In addition, it was also shown to contain nuclear localization signals (1, 2). A quite variable N-terminal domain includes a transcriptional activation function with some receptors.Although ligands have not been identified for orphan nuclear receptors, a variety of results indicate that they have important functions. The simplest is that knockout mutations of these orphans in mice frequently have shown much more dramatic defects than similar mutations of the conventional receptor genes (5-7). We have recently reported an unusual orphan member of the nuclear receptors that contains a ligand binding domain but lacks the conserved DBD (8). This orphan receptor interacts, both in vitro and in the yeast two-hybrid system wi...

Our results suggest that cyclin D1 overexpression may be an early event in hepatocarcinogenesis and that it plays a role in tumor differentiation. In addition, cyclin D1 expression is not correlated with tumor cell proliferation in HCCs.

The aim of this study was to analyze stress distribution and displacement of the maxilla and teeth according to different designs of bone-borne palatal expanders using micro-implants. A three-dimensional (3D) finite-element (FE) model of the craniofacial bones and maxillary teeth was obtained. Four designs of rapid maxillary expanders: one with micro-implants placed lateral to mid-palatal suture (type 1), the second at the palatal slope (type 2), the third as in type 1 with additional conventional Hyrax arms (type 3), and the fourth surgically assisted tooth-borne expander (type 4) were added to the FE models. Expanders were activated transversely for 0.25mm. Geometric nonlinear theory was applied to evaluate Von-Mises Stress distribution and displacement. All types exhibited downward displacement and demonstrated more horizontal movement in the posterior area. Type 3 showed the most transverse displacement. The rotational movement of dentoalveolar unit was larger in types 1 and 3, whereas it was relatively parallel in types 2 and 4. The stresses were concentrated around the micro-implants in types 1 and 3 only. Type 2 had the least stress concentrations around the anchorage and showed alveolar expansion without buccal inclination. It is recommended to apply temporary anchorage devices to the palatal slopes to support expanders for efficient treatment of maxillary transverse deficiency.