Shallow whole-genome sequencing of 13 mice injected with renal cancer cells
ABSTRACT: In order to discover molecular pathways and players in renal cancer development and metastasis, we developed a mouse model to generate sequentially more aggressive and specialized cell lines. Multiple cell lines for primary tumor growth, survival in the blood circulation and lung metastasis or metastatic spread from the primary tumor were generated and analyzed using a multi-layered approach which include large-scale transcriptome, genome and methylome analysis. These are the DNA whole genome sequencing data.
Project description:We report that the H2B deubiquitinating enzymes USP22, USP27x and USP51 have both unique and overlapping target loci. Comparing H2Bb1 (K120) distribution profiles of MCF7 cells stably expressing shRNA targeting ATXN7L3, USP22, USP27X or USP51 and cells expressing non-targeting shRNA.
Project description:In vivo high frequency stimulated of left dentate gyrus was performed on anaesthetised rats followed by RNA-seq to study long-term potentiation. Both left and right dentate gyrus was collected, sequenced and compared against each other for naive rats and for rats 30 min, 2 hours, and 5 hours post-HFS.
Project description:RA signalling regulated endothelial to hematopoietic transition and HSC generation. EB- or FL-derived HSPC were profiled before (d0) or after (d6) 6 days of treatment with 0.2uM AM580 on OP9, and after 6 additional days of expandion of OP9 (d12) without treatment.
Project description:Purpose: We wanted to know how histone variants H3.3 and H2AZ are deposited into genes and enhancers during gene activation Methods: U2OS cells were harvested at 70% confluency with formaldehyde crosslinking for ChIP-seq without crosslinking for RNA-seq. ChIP DNA were purified through standard chromatin immunoprecipitation using an Pol II, MED26, EP400, H3.3, H2AZ, H3k4me1 and K3K18ac antibodies. Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 platform. Total RNA was extracted with Trizol, digested with DNaseI and further purified by acid phenol. Libraries were prepared with Illumina TruSeq RNA Sample Prep Kits v2 and were sequenced on Illumina HiSeq 2000. Conclusion: Our biochemical and genomics (ChIP-Seq and mRNA-Seq) data show that EP400 contributes to H3.3 deposition in significantly with less of an effect on H2AZ in both genes and enhancers Enrichment of EP400, Pol II, MED26, Histone varinat H3.3 and H2AZ, and enhancers marks H3K4me1 and H3K18ac in either Mock and/or EP400 knockdown conditions on chromatin were generated by ChIP-Seq. mRNA profiles under Mock siRNA or EP400siRNA were generated by deep sequencing, using Illumina HiSeq 2000.
Project description:HOXA7 regulates FL-HSPC self-renewal in vitro and in vivo. We profiled EB-HSPCs after HOXA7 overexpression (EB-HOXA7), or with a control vector (EB-CTR), to assess the gene expression programs regulated by HOXA7. CD34+CD38-CD43+CD90+ HSPCs were infected with lentiviral FUGW vector either empty (FUGW-GFP) or encoding HOXA7(FUGW-GFP-HOXA7) protein. Cells were expanded on op9 for 15 days and than sorted for GFP HSPC immunophenotype.
Project description:To study the epigenetic regulation of intestinal epithelium we focus on the role of chromatin modulators. Lysine-specific histone demethylase 1a (KDM1A, LSD1) is one of the enzymes that can erase the H3K4me1/2 mark. To assess the role of LSD1 in intestinal epithelium we studied wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. We found that KO mice completely lack Paneth cells, and have altered stem cell characteristics compared to WT littermates. To assess genome-wide ATAC levels in WT and KO small intestines, we isolated intestinal epithelium tissue from wild type mice and LSD1 KO mice. This tissue was digested to single cells and performed ATAC seq as described in the protocols.
Project description:The combination of chemotherapy and RNA interference is a promising approach for efficient cancer therapy. However, the success of such a strategy is hampered by the lack of suitable vectors to coordinate small interfering RNA (siRNA) and chemotherapeutic drug into one single platform. We herein report a novel triple-layered pH-responsive micelleplex loading siRNA and alkylated cisplatin prodrug for NF-Kappa B targeted treatment of metastatic breast cancer. The micelles were self-assembled from poly(ethylene glycol)-block-poly(aminolated glycidyl methacrylate)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-b-PAGA-b-PDPA) triblock copolymers. At pH 7.4, the cisplatin prodrug was encapsulated in the hydrophobic PDPA core and siRNA was loaded on the positively charged PAGA interlayer to form the micelleplexes. The PEG corona can prevent protein absorption during blood circulation, minimize non-specific interaction with the reticuloendothelial system, and prolong the systemic circulation of the micelleplexes. The positively charged PAGA interlayer can facilitate deep tumor penetration of the micelleplexes, which, upon cellular uptake, are dissociated in the early endosomes to release anticancer drug payload due to protonation of the PDPA core. Using a 4T1 breast cancer model, we demonstrate that this novel micelleplex co-loaded with cisplatin prodrug and siRNA-p65 is able to simultaneously inhibit tumor growth and suppress distant metastasis of the cancer cells by downregulating NF-kappa B expression. The results reported in this study suggest that siRNA and anticancer drug co-delivery using pH-responsive micelleplexes is a promising strategy for efficient treatment of metastatic cancer.
Project description:Accumulation of intravenously injected cytotoxic liposomes in the skin induces serious toxicity. We used single time point and longitudinal intravital microscopy to understand skin accumulation dynamics of non-PEGylated and PEGylated liposomes after systemic injection into mice. Non-PEGylated egg phosphatidylcholine (PC) liposomes showed short circulation half-life (1.3 h) and immediate aggregation in the blood, with some aggregates lodging in skin microvasculature soon after the injection. At 24 h, and more prominently at 48 h postinjection, liposomes appeared in dermal and subdermal cells. PEGylated egg PC liposomes showed long circulation half-life (22 h) and no aggregation in the blood. PEGylated liposomes started to accumulate in the skin microvasculature as soon as 5 min after the injection. Within 3 h postinjection, PEGylated liposomes accumulated in extravascular cells in the dermis and subdermis. Liposomes were present in the skin for at least 7 days postinjection. A regulatory approved PEGylated liposomal doxorubicin (LipoDox) and empty liposomes of the same composition as LipoDox showed similar skin distribution as PEGylated egg PC liposomes, suggesting that this phenomenon is relevant to liposomes of different lipid composition. Decorating liposomes with shorter PEGs (350 or 700) in addition to PEG 2000 did not decrease the deposition. Outside the capillaries, liposomes partially colocalized with CD45-, F4/80+ cells. The accumulation of liposomes was not due to prior neutrophil/platelet binding and transport across endothelium. Moreover, our studies have excluded a role of complement in the skin accumulation of liposomes. Further understanding of mechanisms of this important phenomenon can improve the safety of liposomal nanocarriers.
Project description:Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5-0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7-10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20-26% of the liposome-entrapped enzyme was found in the liver of which 60-69% was in the lysosomal fraction. Spleen contained 3.6-5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed.