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Mammalian Cell Microinjection Assay to Study the Function of Rac and Rho

Microinjection has been widely used as a technique to introduce proteins and DNA into mammalian cells. A major advantage of mrcroinjection over transfection approaches IS that tt is possible to analyze very early responses to proteins; responses to microinjected proteins can be detect ...

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Cell-Free Assay System for Ras- and Rap1-Dependent Activation of MAP-Kinase Cascade

It is well-established that Ras activates the mitogen-activated protein (MAP) kinase cascade consisting of MAP kinase, extracellular signal-regulated kinase (ERK), ERK kinase (MEK), and MEK kinase in mammals (for reviews, see refs. 1–4). MAP kinase is phosphorylated at both serme/thre ...

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Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipases C and D

Cell-surface receptors regulate hydrolysis of cellular phospholipids that are catalyzed by different classes of phosphohpases having distinct specificities. Depending on cell-type and stimulus, multiple lipid signaling pathways are recruited to allow for the physiolog ...

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Two-Hybrid Analysis of RasRaf Interactions

The yeast two-hybrid system (1) is a genetic method that enables the experimentor to determine whether two proteins can form complexes within yeast cells. The method comprises expressing the proteins of interest as “hybrid” proteins, one fused to a DNA-binding domain and the other protein fus ...

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Cloning and Mutational Analysis of the Shc-Phosphotyrosine Interaction/Phosphotyrosine-Binding Domain

The interaction between tyrosme phosphorylated growth-factor receptors and Src Homology 2 (SH2)-domain-containing proteins plays a critical role in growth-factor mediated signal transduction (1). The SH2 domains bind to receptors at phosphotyrosine residues with specif ...

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Use of Fluorescence Spectroscopy to Study the Regulation of Small G Proteins

The Ras-like low-molecular-weight guanosine triphosphate (GTP)-binding proteins form a superfamily whose members participate in a variety of biological pathways, including the regulation of cell growth and differentiation, vesicular transport, and cytoskeletal organ ...

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Prenylation Assays for Small GTPases

Protein prenylation involves the covalent attachment of Cl5 farnesyl or C20 geranylgeranyl (GG) isoprenoids to cysteine resrdues located at or near the COOH-terminus of various proteins. The majority of cellular proteins that are known to undergo this modtfrcation are small guanos ...

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Analysis of Myristoylated and Palmitoylated Src Family Proteins

Several hundred viral and cellular proteins have been shown to be covalently modified by fatty acids (1–3). The two most common modifications, myristoylatron and palmitoylatron, differ with respect to the type and chemical nature of fatty acid attachment to the polypeptide backbone. Most ...

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Clonal Analyses and Cryopreservation of Neural Stem Cell Cultures

The discovery of stem cell populations in the adult central nervous system (CNS) that continually produce neurons and glial cells, and the hypothesis that they could contribute to neural plasticity/repair, has opened new and exciting areas of research in basic cell biology and regenerati ...

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Culturing Olfactory Ensheathing Cells from the Mouse Olfactory Epithelium

Olfactory ensheathing cells (OECs) are not a class of stem cell, but they are a specialized and highly plastic glial cell that can continuously support the neurogenesis and axonal regeneration of olfactory receptor neurons. Because of this, they have been transplanted into sites of spinal co ...

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Olfactory Ensheathing Cells: Isolation and Culture from the Neonatal Olfactory Bulb

Olfactory ensheathing cells (OECs), which are glia from the olfactory system, have evolved as attractive candidates for transplant-mediated repair based on long-standing knowledge that the olfactory system is one of the only central nervous system tissues that can support neuroge ...

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Adult Neural Stem Cells

Neural stem cells (NSCs) have been identified in the mature central nervous system (CNS), and they reside in specific areas. Cultures of NSCs can be successfully established in vitro by exploiting the NeuroSphere assay. This methodology relies on the continuous exposure of neural cells to mi ...

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Adult Rodent Spinal Cord-Derived Neural Stem Cells: Isolation and Characterization

Self-renewing multipotent stem/progenitor cells have been isolated from various areas of the adult central nervous system, both neurogenic and non-neurogenic, in vitro by using a variety of mitogens and culture conditions. Mitogenic growth factors stimulate the proliferation of ...

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Isolation of Neural Stem and Precursor Cells from Rodent Tissue

Isolation and characterization of neural stem cells and lineage-specific progenitors provide important information for central nervous system development study and regenerative medicine. We describe methods for dissection of rodent embryonic spinal cords by enzymatic s ...

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Generating Neurons from Stem Cells

Recent work shows that major developmental and clinical processes such as central nervous system regeneration and carcinogenesis involve stem cells (SCs) in the brain. In spite of this importance, the requirements of these SCs and their differentiated offspring (neurons, astrocyt ...

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Neural Differentiation of Human Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent and capable of indefinite self-renewal in vitro. These features make them a highly advantageous source for deriving any cell type of the central and peripheral nervous system. We describe neural induction of human (h)ESCs, by using the bone morpho ...

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Nuclear Transfer to Study the Nuclear Reprogramming of Human Stem Cells

Research of stem cells will enable us to understand the development and function of tissues and organs in mammals. The ability to induce regeneration of new tissues from embryonic stem (ES) cells derived from cloned blastocysts via nuclear transfer can be expected in the not-too-distant futu ...

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Production of Neurospheres from CNS Tissue

The relatively recent discovery of persistent adult neurogenesis has led to the experimental isolation and characterization of central nervous system neural stem cell populations. Protocols for in vitro analysis and expansion of neural stem cells are crucial for understanding t ...

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Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood

The bone marrow is clearly much more than a reservoir of stem cells that repopulates blood cell lineages throughout life. The marrow also contains nonhematopoietic stem cells, which are much more versatile than previously appreciated. These nonhematopoietic stem/progenitor cells ...

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Production and Characterization of Immortal Human Neural Stem Cell Line with Multipotent Differentiation Property

We document the protocols and methods for the production of immortalized cell lines of human neural stem cells from the human fetal central nervous system (CNS) cells by using a retroviral vector encoding v-myc oncogene. One of the human neural stem cell lines (HB1.F3) was found to express nestin and ...

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