Project description:We report the application of RNA-Seq technology for transcriptome analysis on Pdx1 + Ngn3 co-transfected bmMSCs. Bioinformatics analyses indicated that clean reads of the two libraries accounted for 97.24% and 97.97% of the total reads, which indicated high quality of the raw datasets. Total 2325 differentially expressed genes (DEGs) were identified in reprogrammed bmMSCs compared to the negative control, including 1476 genes up-regulated and 849 genes down-regulated. The RNA-Seq analysis globally validated in the reprogrammed cells the expression of a large number of genes involved in pancreas development, endocrine specification and islet differentiation. To the best of our knowledge, this is the first application of RNA-Seq analysis on reprogrammed MSCs towards pancreatic cells.
Project description:In order to establish a rat embryonic stem cell transcriptome, mRNA from rESC cell line DAc8, the first male germline competent rat ESC line to be described and the first to be used to generate a knockout rat model was characterized using RNA sequencing (RNA-seq) analysis.
Project description:Analysis of LBNF1 rat testes from controls, containing both somatic and all germ cell types and from irradiated rats in which all cells germ cells except type A spermatgogonia are eliminated. Results provide insight into distinguishing germ and somatic cell genes and identification of somatic cell genes that are upregulated after irradiation.
Project description:During pancreatic development, Neurogenin3 (Ngn3) promotes the differentiation of endocrine cells, including insulin-producing β-cells. Our previous work has shown that Ngn3 phosphorylation on multiple serine-proline (SP) sites increases protein stability and endocrine differentiation (Azzarelli et al., DevCell 2017). Here, we aim to exploit our recent data to investigate whether manipulation of Ngn3 phosphostatus might improve in vitro generation of β-cells for replacement therapies in diabetes. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of 3 transcription factors, Ngn3, Pdx1 and MafA. Using this approach we show that pancreatic ductal cells grown in vitro as 3D organoids can be reprogrammed to express insulin. The efficiency can be strongly enhanced by substituting wild type Ngn3 with a the un(der)phosphorylated and more active Ngn3 form (6S-A Ngn3). Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type or phosphomutant Ngn3, alone or in various combination Pdx1 and/or MafA. The analysis revealed endocrine differentiation and in particular β-like cell gene expression in the conditions where Ngn3 was in combination with both Pdx1 and MafA.
Project description:NGN3 is a transcription factor whose transient expression during pancreatic development is vital for the generation of endocrine pancreatic cells, including beta cells. NGN3 stabilisation has been shown to induce exocrine-to-endocrine cell plasticity in the murine pancreas, making it a viable target for therapies aiming to replenish beta cells after immune-mediated destruction in type 1 diabetes patients. Here, we set out to identify new interactors of NGN3 that could play a role in its post-translational regulation. We transfected HEK293A cells with HA-tagged NGN3 and carried out immunoprecipitation of the HA-tag, followed by analysis of co-immunoprecipitated interactors via LC-MS/MS.
Project description:Induced overexpression of Pdx1 in activin-induced endoderm population resulted in the upregulation of pancreas-related genes such as insulin 1 and 2 at day 20. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we next expressed neurogenin3 (Ngn3) together with Pdx-1. Induced overexpression of Pdx1 together with Ngn3 dramatically increased Insulin 1 mRNA by day 9 differentiation. The levels of insulin 1 mRNA present in the induced EBs represented approximately 100 % of that found in insulinoma cell line, betaTC6. We also confirmed insulin and C-peptide staining by immunohistochemistry. These cells process and secrete insulin and respond to various insulin secretagogues. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions at the level of pancreatic specification of endoderm in this model. Microarray analysis showed that Pdx1/Ngn3 regulated the expression of a broad spectrum of pancreatic endocrine cell-related genes.
Project description:Living organisms are intricate systems with dynamic internal processes. Their RNA, protein, and metabolite levels fluctuate in response to variations in health and environmental conditions. Among these, RNA expression is particularly accessible for comprehensive analysis, thanks to the evolution of high throughput sequencing technologies in recent years. This progress has enabled researchers to identify unique RNA patterns associated with various diseases, as well as to develop predictive and prognostic biomarkers for therapy response. Such cross-sectional studies allow for the identification of differentially expressed genes (DEGs) between groups, but they have limitations. Specifically, they often fail to capture the temporal changes in gene expression following individual perturbations and may lead to significant false discoveries due to inherent noise in RNA sequencing sample preparation and data collection. To address these challenges, our study hypothesized that frequent, longitudinal RNA sequencing (RNAseq) analysis of blood samples could offer a more profound understanding of the temporal dynamics of gene expression in response to drug interventions, while also enhancing the accuracy of identifying genes influenced by these drugs. In this research, we conducted RNAseq on 829 blood samples collected from 84 Sprague-Dawley lab rats. Excluding the control group, each rat was administered one of four different compounds known for liver toxicity: tetracycline, isoniazid, valproate, and carbon tetrachloride. We developed specialized bioinformatics tools to pinpoint genes that exhibit temporal variation in response to these treatments.
Project description:Induced overexpression of Pdx1 in activin-induced endoderm population resulted in the upregulation of pancreas-related genes such as insulin 1 and 2 at day 20. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we next expressed neurogenin3 (Ngn3) together with Pdx-1. Induced overexpression of Pdx1 together with Ngn3 dramatically increased Insulin 1 mRNA by day 9 differentiation. The levels of insulin 1 mRNA present in the induced EBs represented approximately 100 % of that found in insulinoma cell line, betaTC6. We also confirmed insulin and C-peptide staining by immunohistochemistry. These cells process and secrete insulin and respond to various insulin secretagogues. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions at the level of pancreatic specification of endoderm in this model. Microarray analysis showed that Pdx1/Ngn3 regulated the expression of a broad spectrum of pancreatic endocrine cell-related genes. On day 4 of a multiday differentiation protocol, EBs were dissociated and cultured in serum-free growth medium supplemented with differentiation facots, with or without Dox (1 ug/ml). On day 6, EBs were replated on gelatin in 12-well low-cluster dishes (Nunc) to obtain non-adherent floating EBs with or without Dox (1 ug/ml) and cultured out to day 13, at which time RNA was harvested for microarray analysis.