Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Direct Lineage Conversion of Pancreatic Exocrine to Endocrine Beta Cells In Vivo with Defined Factors
Pancreatic exocrine cells can be directly converted to insulin+ beta cells by adenoviral-mediated expression of three transcription factors Pdx1, Mafa, and Ngn3 in the adult mouse pancreas (Zhou et al., Nature 455(7213):627–632, 2008). This direct reprogramming approach offers a strategy to replenish beta-cell mass and may be further developed as a potential future treatment for diabetes. Here, we provide a detailed protocol for inducing exocrine to beta-cell reprogramming in mice. We also describe key analyses we routinely use to assess the phenotype and function of reprogrammed cells. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Transdifferentiation of Mouse Fibroblasts and Hepatocytes to Functional Neurons
Nuclear reprogramming by defined transcription factors became of broad interest in 2006 with the work of Takahashi and Yamanaka (Cell 126:663–676, 2006), but the first example of cell fate reshaping via ectopic expression of transcription factor was provided back in 1987 when Davis and colleagues induced features of a muscle cell in fibroblast using the muscle transcription factor MyoD (Davis et al., Cell 51:987–1000, 1987). In 2010 our laboratory described how forced expression of the three neuronal transcription factors Ascl1, Brn2, and Myt1l rapidly converts mouse fibroblasts into neuronal cells that exhibit...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Generation of Induced Pluripotent Stem Cells Using Chemical Inhibition and Three Transcription Factors
Generation of induced pluripotent stem (iPS) cells from differentiated cells has traditionally been performed by overexpressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. However, inclusion of c-Myc in the reprogramming cocktail can lead to expansion of transformed cells that are not fully reprogrammed, and studies have demonstrated that c-Myc reactivation increases tumorigenicity in chimeras and progeny mice. Moreover, chemical inhibition of Wnt signaling has been shown to enhance reprogramming efficiency. Here, we describe a modified protocol for generating iPS cells from murine fibroblasts using chemical i...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Conversion of Epiblast Stem Cells to Embryonic Stem Cells Using Growth Factors and Small Molecule Inhibitors
Stem cell in vitro culture is a useful model system to study mechanisms underlying transitions between defined cell states. Epiblast stem cells, in addition to being capable of somatic differentiation, can be converted to a more primitive embryonic stem cell-like state, by overexpression of specific transcription factors. Here, we describe a reliable method to accomplish—and potentially further study—the transgene-independent reversion from epiblast stem cells to ES cells using administration of specific growth factors and small molecule inhibitors. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Derivation and Manipulation of Trophoblast Stem Cells from Mouse Blastocysts
The trophoblast is the first lineage to undergo differentiation during mammalian development. In the preimplantation blastocyst embryo, two cell types are present including the inner cell mass (ICM) and the trophectoderm (TE). ICM cells exhibit pluripotent potential, or the capacity to give rise to all cells represented in the adult organism, while TE cells are multipotent and are therefore only capable of differentiating into trophoblast lineages represented in the placenta. The TE is essential for implantation of the embryo into the uterine tissue, formation of trophoblast lineages represented in the placenta, and exchan...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
In Vitro Maturation and In Vitro Fertilization of Mouse Oocytes and Preimplantation Embryo Culture
Epigenetic regulation of gene expression in the germline is important for reproductive success of mammals. Misregulation of genes whose expression is correlated with reproductive success may result in subfertility or infertility. To study epigenetic events that occur during oocyte maturation and preimplantation embryo development, it is important to generate large numbers of ovarian follicles and embryos. Oocyte maturation can be modeled using in vitro maturation (IVM), which is a system of maturing ovarian follicles in a culture dish. In addition, fertilization and early embryogenesis can be modeled using in vitro fertili...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Correlating Histone Modification Patterns with Gene Expression Data During Hematopoiesis
Hematopoietic stem cells (HSC) in mammals are an ideal system to study differentiation. While transcription factors (TFs) control the differentiation of HSCs to distinctive terminal blood cells, accumulating evidence suggests that chromatin structure and modifications constitute another critical layer of gene regulation. Recent genome-wide studies based on next-generation sequencing reveal that histone modifications are linked to gene expression and contribute to hematopoiesis. Here, we briefly review the bioinformatics aspects for ChIP-Seq and RNA-Seq data analysis with applications to the epigenetic studies of hematopoie...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Use of Genome-Wide RNAi Screens to Identify Regulators of Embryonic Stem Cell Pluripotency and Self-Renewal
Embryonic stem cells (ESCs) are characterized by two defining features: pluripotency and self-renewal. They hold tremendous promise for both basic research and regenerative medicine. To fully realize their potentials, it is important to understand the molecular mechanisms regulating ESC pluripotency and self-renewal. The development of RNA interference (RNAi) technology has revolutionized functional genetic studies in mammalian cells. In recent years, genome-wide RNAi screens have been adopted to systematically study ESC biology, and have uncovered many previously unknown regulators, including transcription factors, chroma...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
A Description of the Molecular Signatures Database (MSigDB) Web Site
Annotated lists of genes help researchers to prioritize their own lists of candidate genes and to plan follow-up studies. The Molecular Signatures Database (MSigDB) is one of the most widely used knowledge base repositories of annotated sets of genes involved in biochemical pathways, signaling cascades, expression profiles from research publications, and other biological concepts. Here we provide an overview of MSigDB and its online analytical tools. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Interpreting and Visualizing ChIP-seq Data with the seqMINER Software
Chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq) is a common method to study in vivo protein–DNA interactions at the genome-wide level. The processing, analysis, and biological interpretation of gigabyte datasets, generated by several ChIP-seq runs, is a challenging task for biologists. The seqMINER platform has been designed to handle, compare, and visualize different sequencing datasets in a user-friendly way. Different analysis methods are applied to understand common and specific binding patterns of single or multiple datasets to answer complex biological questions. Here, we give a deta...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Visualization and Clustering of High-Dimensional Transcriptome Data Using GATE
This article provides a brief guide to using GATE effectively. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Identifying Stem Cell Gene Expression Patterns and Phenotypic Networks with AutoSOME
Stem cells have the unique property of differentiation and self-renewal and play critical roles in normal development, tissue repair, and disease. To promote systems-wide analysis of cells and tissues, we developed AutoSOME, a machine-learning method for identifying coordinated gene expression patterns and correlated cellular phenotypes in whole-transcriptome data, without prior knowledge of cluster number or structure. Here, we present a facile primer demonstrating the use of AutoSOME for identification and characterization of stem cell gene expression signatures and for visualization of transcriptome networks using Cytos...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Spatial Clustering for Identification of ChIP-Enriched Regions (SICER) to Map Regions of Histone Methylation Patterns in Embryonic Stem Cells
Chromatin states are the key to embryonic stem cell pluripotency and differentiation. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-Seq) is increasingly used to map chromatin states and to functionally annotate the genome. Many ChIP-Seq profiles, especially those of histone methylations, are noisy and diffuse. Here we describe SICER (Zang et al., Bioinformatics 25(15):1952–1958, 2009), an algorithm specifically designed to identify disperse ChIP-enriched regions with high sensitivity and specificity. This algorithm has found a lot of applications in epigenomic studies. In this Chap...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Use Model-Based Analysis of ChIP-Seq (MACS) to Analyze Short Reads Generated by Sequencing Protein–DNA Interactions in Embryonic Stem Cells
Model-based Analysis of ChIP-Seq (MACS) is a computational algorithm for identifying genome-wide protein–DNA interaction from ChIP-Seq data. MACS combines multiple modules to process aligned ChIP-Seq reads for either transcription factor or histone modification by removing redundant reads, estimating fragment length, building signal profile, calculating peak enrichment, and refining and reporting peak calls. In this protocol, we provide a detailed demonstration of how to apply MACS to analyze ChIP-Seq datasets related to protein–DNA interactions in embryonic stem cells (ES cells). Instruction on how to interpre...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Analysis of Next-Generation Sequencing Data Using Galaxy
The extraordinary throughput of next-generation sequencing (NGS) technology is outpacing our ability to analyze and interpret the data. This chapter will focus on practical informatics methods, strategies, and software tools for transforming NGS data into usable information through the use of a web-based platform, Galaxy. The Galaxy interface is explored through several different types of example analyses. Instructions for running one’s own Galaxy server on local hardware or on cloud computing resources are provided. Installing new tools into a personal Galaxy instance is also demonstrated. (Source: Springer protocol...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
