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Single-Cell PCR Methods for Studying Stem Cells and Progenitors

Knowledge of the molecular and cellular events characterizing osteoblast development is growing as new markers, including important classes of regulatory molecules such as transcription factors (e.g., Cbfa-1 ), are elucidated. Nevertheless, a paucity of definitive and specific ...

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The Use of Chemically Defined Media for the Analyses of Early Development in ES Cells and Mouse Embryos

During embryonic development, primitive ectoderm forms three primary germ layers, the mesoderm, the ectoderm, and the endoderm. These germ layers interact forming all the tissues and organs of the developing embryo. The influences controlling the transition of ectoderm to visceral a ...

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Nonradioactive Labeling and Detection of mRNAs Hybridized onto Nucleic Acid cDNA Arrays

Cellular gene expression changes during ontogenetic development of cell physiological activation-inhibtion and differentiation. Classical molecular assays like Southern, Northern, or Western blotting display only a few genes at once. The analysis of complex alterations ...

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Expression Profiling Using Quantitative Hybridization on Macroarrays

Expression studies performed on a genome scale have become very popular and provide an important link between the sequence and the function of a specific gene, thereby constituting the first step towards the elucidation of the function of specific genes. By correlating the modulation of gene ...

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Isolation of Antigen-Specific Intracellular Antibody Fragments as Single Chain Fv for Use in Mammalian Cells

The control of gene function by the modulation of mRNA translation, stability, or protein activity has important implications for the treatment of disease, as well as in research programs designed to study the biological role of proteins. For instance, preventing protein function in spec ...

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Detection and Visualization of Protein Interactions with Protein Fragment Complementation Assays

A first step in defining the function of a novel gene is to determine its interactions with other gene products in an appropriate context; that is, because proteins make specific interactions with other proteins as part of functional assemblies, an appropriate way to examine the function of the p ...

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Screening for Protein-Protein Interactions in the Yeast Two-Hybrid System in Embryonic Stem Cells

The two-hybrid system (THS) (1) is a molecular genetic screen that detects protein-protein interactions. The protein specified by the yeast GAL4 gene activates the transcription of genes involved in galactose metabolism. It has two functional domains, a DNA binding domain, Gal4BD, and a tr ...

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Direct Selection of cDNAs by Phage Display

Dozens of genomes will be partly or completely sequenced over the next few years. In the post genomic area, we are now facing the challenge of functionally characterizing thousands of genes generated by the genome projects. Selective enrichment of clones encoding a desired gene product and ra ...

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Analysis of the Cell Cycle in Mouse Embryonic Stem Cells

The molecular mechanisms underlying self-renewal of pluripotent embryonic stem (ES) cells is still poorly understood. Deciphering these mechanisms is of prime importance for at least two reasons: (1) ES cells derive from, and are closely related to, the pluripotent stem cells of the blast ...

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Murine Embryonic Stem Cells as a Model for Stress Proteins and Apoptosis During Differentiation

The early phase of murine embryonic stem (ES) cell differentiation is accompanied with the expression of proteins that are key players in this phenomenon. The function of some these proteins can be analyzed by using the classical “knock-out gene technology”, that consists of the inactivati ...

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Effects of Altered Gene Expression on ES Cell Differentiation

Little is known about the molecular mechanisms controlling the earliest cellular differentiation events of the mammalian embryo. Pluripotent embryonic stem (ES) cell lines, derived from cells of the inner cell mass of the blastocyst, are an important experimental system that can be used ...

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Regulation of Gap Junction Protein (Connexin) Genes and Function in Differentiating ES Cells

Gap junctions are specialized cell-cell junctions that directly link the cytoplasm of neighboring cells. They mediate the direct transfer of low molecular weight (

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Hypoxic Gene Regulation in Differentiating ES Cells

Mammalian development occurs in the hypoxic environment of the uterus (1,2). Initially, the limited oxygen available can adequately diffuse to all the cells of the growing conceptus. However, with continued growth, diffusion becomes less efficient and results in a “physiologic hypox ...

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Embryonic Stem Cell Differentiation as a Model to Study Hematopoietic and Endothelial Cell Development

With the attractive potential therapeutic uses of both endothelial and hematopoietic cells in medicine, much attention has been focused upon the differentiation of embryonic stem (ES) cells into mature cells of these lineages. In this chapter, we present a culture system that has success ...

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Analysis of Bcr-Abl Function Using an In Vitro Embryonic Stem Cell Differentiation System

Numerous causative genes involved in human cancers, such as leukemias and lymphomas, have been identified (1). Considerable evidence has accumulated showing that disruptions of these genes can affect hematopoietic cell differentiation and growth. However, it remains largely un ...

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The GTPase Cycle How Dominant Inhibitory Mutants Block the Biological Functions of Small GTPases

There are two key methods which yield information about the cellular function of a protein. One can introduce into cells or whole organisms a form of the protein that is constitutively active, and which therefore functions independent of the normal regulatory mechanisms. This technique has ...

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Preparation of GTPases for Structural and Biophysical Analysis

Members of the Ras superfamily of small GTPases (p21) are involved in the regulation of a large variety of key cellular processes, including cell differentiation and proliferation, membrane trafficking, and nuclear import and export. Based on sequence homology, this superfamily can be d ...

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Fluorescence Methods in the Study of Small GTP-Binding Proteins

A great variety of small guanosine triphosphate (GTP)-binding proteins are known, and each of these in turn interacts with a variety of regulatory proteins, including guanine-nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs) and guaninine nucleo ...

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Using cDNA-Representational Difference Analysis (cDNA-RDA) in Combination with Microarrays to Identify Rac Regulated Genes

Small GTPases of the Ras superfamily are molecular switches which cycle between an active guanosine triphosphate (GTP)-bound and an inactive guanosine diphosphate (GDP)-bound state. They integrate signals from the cell surface to the nucleus, regulating important cellular acti ...

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Effects of Rho Family GTPases on Cell-Cell Adhesion

Cell-cell adhesion is dynamically rearranged in various cellular processes, including cell scattering, wound healing, developmental morphogenesis, and tumor metastasis. Cadherins are Ca2+-dependent homophilic cell-cell adhesion molecules (1) and bind β-catenin or pla ...

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