The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.
Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2 , EXO1 , and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.
BackgroundNucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells.ResultsWe generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs’ tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs.ConclusionsThe re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0109-x) contains supplementary material, which is available to authorized users.
Digital light processing (DLP)-based 3D printing technique holds promise in fabricating scaffolds with high precision. Here raw calcium phosphate (CaP) powders were modified by 5.5% monoalcohol ethoxylate phosphate (MAEP) to ensure high solid loading and low viscosity. The rheological tests found that photocurable slurries composed of 50 wt % modified CaP powders and 2 wt % toners were suitable for DLP printing. Based on geometric models designed by CAD system, three printed CaP ceramics with distinct macroporous structures were prepared, including simple cube, octet-truss, and inverse face-centered cube (fcc), which presented the similar phase composition and microstructure, but the different macropore geometries. Inverse-fcc group showed the highest porosity and compressive strength. The in vitro and in vivo biological evaluations were performed to compare the bioactivity of three printed CaP ceramics, and the traditional foamed ceramic was used as control. It suggested that all CaP ceramics exhibited good biocompatibility, as evidence by an even bone-like apatite layer formation on the surface, and the good cell proliferation and spreading. A mouse intramuscular implantation model found that all of CaP ceramics could induce ectopic bone formation, and Foam group had the strongest osteoinduction, followed by Inverse-fcc, while Cube and Octet-truss had the weakest one. It indicated that macropore geometry was of great importance to affect the osteoinductivity of scaffolds, and spherical, concave macropores facilitated osteogenesis. These findings provide a strategy to design and fabricate high-performance orthopedic grafts with proper pore geometry and desired biological performance via DLP-based 3D printing technique.
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