bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immortalizing Central Nervous System Cells
Authors: Whittemore Scott R. 
Published: June 2004

AbstractThis unit presents methods for isolating clonal, neural‐derived cell lines. One approach for isolating such neural cell lines involves a replication‐deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.
Isolation and Purification of Primary Schwann Cells
Authors: Weinstein David E. , Wu Rina 
Published: June 2004

AbstractSchwann cells are the glial cells of the peripheral nervous system (PNS) which both myelinate and provide trophic support for their associated axons. Their functions are critical for proper development, homeostasis, and regeneration of the PNS. Schwann cells can be isolated and expanded in culture. Such a culture can be used as a source of myelinating glia in culture or as a source of Schwann cells for various cell and molecular biological experiments. The proliferative ability of these cells is exploited in the isolation procedures presented here.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Gyuris Jeno , Brent Roger 
Published: June 2004

AbstractThe parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two‐hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.
Selection of Transfected Mammalian Cells
Authors: Mortensen Richard , Chestnut Jonathan D. , Hoeffler James P. , Kingston Robert E. 
Published: June 2004

AbstractAnalysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a support protocol.
Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
Authors: Mortensen Richard 
Published: June 2004

AbstractGene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.
Differential Display of mRNA by PCR
Authors: Livesey F.J. , Hunt C.P. 
Published: June 2004

AbstractThis unit outlines the polymerase chain reaction (PCR)‐based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The approach described here is a modification of the original method, referred to as single base‐anchored differential display. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. The support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.
Production of Antipeptide Antisera
Authors: Coligan John E. , Tam James P. , Shao Jun 
Published: June 2004

AbstractThis unit describes methods used for the chemical coupling of synthetic peptides to carrier proteins, required for the preparation of peptide immunogens. The carrier protein described here is keyhole limpet hemocyanin (KLH) because it is the one most commonly used. However, other proteins may be used in place of KLH, including bovine serum albumin (BSA) and ovalbumin. Coupling may be accomplished as described with MBS, which requires a Cys residue in the peptide, or with glutaraldehyde, EDCI, or BDB. Also included are an assay for detecting free sulfhydryl (SH) groups, a means of calculating coupling efficiency, an immunization schedule, an indirect ELISA, and a method for preparing a peptide affinity column. The final methodology described is the multiple antigen (MAP) system.
Preparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings
Authors: Karpiak Veronica C. , Plenz Dietmar 
Published: June 2004

AbstractPreparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings (Veronica C. Karpiak and Dietmar Plenz, National Institutes of Mental Health, NIH, Bethesda, Maryland). Recording from neuronal cultures with multi‐electrode arrays (MEAs) provides a powerful tool for studying neuronal activity with many neurons simultaneously in vitro. This unit describes the detailed steps necessary for growing organotypic cultures on MEAs and the typical neuronal activity that is obtained with this methodology.
Imaging Nervous System Activity with Voltage‐Sensitive Dyes
Authors: Zecevic Dejan , Djurisic Maja , Cohen Lawrence B. , Antic Srdjan , Wachowiak Matt , Falk Chun X. , Zochowski Michal R. 
Published: June 2004

AbstractOptical recording with a voltage‐sensitive dye is advantageous where membrane potential must be recorded in many sites at once. This unit describes methods for making voltage‐sensitive dye measurements on different preparations to study (1) how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and where spikes are initiated; (2) how a nervous system generates a behavior in Aplysia abdominal ganglion; and (3) responses to sensory stimuli and generation of motor output in the vertebrate brain by simultaneous measurement of population signals from many areas. The approach is three‐pronged: (1) find the dye with the largest signal‐to‐noise ratio; (2) reduce extraneous sources of noise; and (3) maximize the number of photons measured to reduce the relative shot noise. A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.
Measurement of NO and NO Synthase
Authors: Weissman Ben Avi , Gross Steven S. 
Published: June 2004

AbstractNitric oxide (NO) is a key biosignaling molecule produced in both peripheral tissues and the central nervous system by a family of enzymes known as nitric oxide synthases (NOSs). NOSs convert L‐arginine to stoichiometric quantities of NO and L‐citrulline using molecular oxygen and NADPH as cofactors. Techniques for measurement of NO and NOS activity are essential to demonstrate the role of NO and NO‐derived species in biological systems. This unit describes two methods for detection of NO: a direct method employing chemiluminescent detection and one based on quantification of the stable oxidation products with detection using the Greiss reagent. Additionally, NOS activity can be quantified by measuring the conversion of radiolabeled L‐arginine to radiolabeled L‐citrulline.
Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues
Authors: Hall Edward D. , Andrus Paula K. 
Published: June 2004

AbstractAn important role for oxygen radical‐mediated neuronal damage has been implicated in a number of acute and chronic neurodegenerative disorders. Particular interest has centered upon oxygen radical‐induced, iron‐catalyzed lipid peroxidation (LP) as the principal mechanism of the neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in methods for monitoring increased oxygen radical levels as an index of oxidative stress as well as markers of LP‐associated oxidative injury in a number of in vitro and in vivo model systems. This unit provides a detailed description of the salicylate trapping method for the measurement of the most highly reactive oxygen radical, the hydroxyl radical, as well as several direct or indirect methods for assessment of cellular LP in either cell cultures or in in vivo models.
Animal Tests of Anxiety
Authors: File Sandra E. , Lippa Arnold S. , Beer Bernard , Lippa Morgen T. 
Published: June 2004

AbstractAnimal tests of anxiety are used to screen novel compounds for anxiolytic or anxiogenic activity, to investigate the neurobiology of anxiety, and to assess the impact of other occurrences such as exposure to predator odors or early rearing experiences. This unit presents protocols for the most commonly used animal tests of anxiety. The Geller‐Seifter conflict test, the social interaction test, light/dark exploration, the elevated plus‐maze, defensive burying, and the thirsty rat conflict. The protocols are described in terms of drug screening tests, but can be modified easily for other purposes.
Assessment of Spatial Memory Using the T Maze
Authors: Wenk Gary L. 
Published: June 2004

AbstractThis unit describes the use of the T maze to assess spatial memory, and takes into account the alternating behavior of rats in searching for food. The task is based on the premise that animals have evolved an optimal strategy to explore their environment and obtain food with a minimum amount of effort. The T maze has been most extensively used to investigate specific aspects of spatial working memory, which is operationally defined as information that is only useful to a rat during the current experience with the task. A modification of the maze allows for the assessment of reference memory, defined as information that is useful across all exposures to the task (i.e., on any day of testing). Finally, in the absence of food‐deprivation, a simple T maze can be used as described to assess spontaneous alternation.
Macronutrient Selection in Experimental Animals
Authors: Kanarek Robin B. 
Published: June 2004

AbstractTo address the question of how animals choose diets from an array of nutritionally different foods, researchers have designed experiments in which animals are provided with separate sources of the three macronutrients, protein, fat and carbohydrate. These experiments are useful for investigating the neuroanatomical and neurochemical mechanisms involved in food choice, as well as for assessing the effects of changes in physiological status (e.g., pregnancy; lactation and increased energy expenditure) or disease states (e.g., diabetes; and obesity) on nutrient intakes.
Rodent Models of Depression: Forced Swimming and Tail Suspension Behavioral Despair Tests in Rats and Mice
Authors: Porsolt Roger D. , Brossard Geneviève , Hautbois Carine , Roux Sylvain 
Published: June 2004

AbstractRodents forced to swim in a narrow space from which there is no escape will, after an initial period of vigorous activity, adopt a characteristic immobile posture, making only those movements necessary to keep their heads above the water. It was hypothesized that immobility reflected the animals' having learned that escape was impossible and their having given up hope. Immobility was therefore given the name “behavioral despair”. Immobility was subsequently found to be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a useful test for screening novel antidepressants in rats and is presented in this unit. An equivalent procedure in the mouse is also described along with a “dry” version of the test where immobility is induced simply by suspending the mouse by the tail.
Fear‐Potentiated Startle in Rats
Authors: Davis Michael 
Published: June 2004

AbstractFear‐potentiated startle measures the increase in the startle reflex elicited by a sudden noise in the presence of a cue that has previously been paired with footshock. It involves an initial training session in which a cue, such as a light, is paired with footshock, and then a later test session in which startle is elicited in the presence or absence of the cue. It thus involves associative learning, memory for that association measured during the retention test, and conditioned fear in the presence of the cue. Drugs that interfere with learning, memory, or fear and anxiety can alter fear‐potentiated startle depending on when they are given with respect to training and testing.
Latent Inhibition
Authors: Weiner Ina 
Published: June 2004

AbstractLatent inhibition (LI) refers to the deleterious effects of nonreinforced stimulus preexposure on its subsequent conditioning. LI is considered to index an organism's ability to ignore inconsequential stimuli and as such provides a useful tool to measure attentional processes as well as their disruption and enhancement by various pharmacological, physiological and behavioral manipulations. During the last decade, LI has received increasing interest as an animal model of schizophrenia and of antipsychotic drug action. This unit describes the measurement of LI in the conditioned emotional response and two‐way active avoidance procedures, including parametric variations which allow the demonstration of the disruption and the potentiation of this phenomenon.
Ultrasonic Vocalizations in Rat and Mouse Pups
Authors: Hofer Myron A. , Shair Harry N. , Brunelli Susan A. 
Published: June 2004

AbstractThe ultrasonic vocalization (USV), or isolation calling response, of infant rats and mice has been studied as a measure of the intensity of an aversive affective state and as an early communicative behavior between pup and mother. The four protocols described in this unit are for the basic isolation testing procedure, and for elicitation of the contact quieting response to littermates and/or dam, the potentiation of isolation calling response by a prior brief maternal interaction and the predator‐induced suppression of USV by the scent of an unfamiliar male. These procedures for the elicitation of USV, and for its regulation by different kinds of social interaction, provides the basis for experimental research on the early development of emotion and communication in an animal model system.
Experimental Autoimmune Encephalomyelitis (EAE)
Authors: Racke Michael K. 
Published: June 2004

AbstractThe procedures described in this unit utilize murine models exclusively since murine EAE often results in a relapsing/remitting disease, similar to the early phase of most MS patients. EAE in the Lewis rat is a monophasic illness in which animals experience a single episode of paralysis from which most recover completely. This unit presents two methods for inducing EAE in mice: active induction and adoptive transfer
Animal Models of Retroviral Encephalopathies: Feline Model
Authors: Podell Michael , Buck Wayne R. , Hayes Kathleen A. , Gavrilin Mikhail A. , Mathes Lawrence E. 
Published: June 2004

AbstractHuman immunodeficiency virus infection in children and adults results in a progressive neurodegenerative disease consistent with a predominant subcortical mediated dementia. Techniques for developing a feline model of the early stages of lentiviral‐associated neurodegeneration are presented. The behavioral, neurophysiologic, immunologic, virologic, and neuropathologic aspects of this model are also described.
Inducing Photochemical Cortical Lesions in Rat Brain
Authors: Bergeron Marcelle 
Published: June 2004

AbstractIn the rat photochemical cortical lesion model described in this unit, an intravascular photochemical reaction induces endothelial damage resulting in platelet aggregation, thrombosis, thrombotic response (secretion of factors by the platelets) and permanent cerebral vascular occlusion. Because thrombosis is produced in pial vessels, the resulting cortical infarct is generally smaller and more reproducible than in the models involving occlusion of the middle cerebral artery. The surgical procedures involved are limited, making this model generally easier to perform and less invasive than most other models of permanent focal ischemia that involve mechanical occlusion of major cerebral arteries.
Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Authors: Hawley‐Nelson Pamela , Ciccarone Valentina 
Published: June 2004

AbstractThe development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipid‐based transfection reagents has accelerated biology research in studies of gene expression, control of cell growth, and cell lineage. In this unit, DNA transfection is described for adherent mammalian cells (both cells and primary cultures) along with an alternate procedure for enhanced transfection. A protocol is also described for transfection of suspension cells (lymphoid, myeloid and leukemic‐derived cells). In addition, transfection of RNA into adherent mammalian cells and DNA transfection into insect cells are presented. Importantly, a detailed procedure is described for optimization of reagents and transfection conditions.
Preparation of Bacterial Plasmid DNA
Authors: Engebrecht JoAnne , Heilig J.S. , Brent Roger 
Published: June 2004

AbstractThe protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion‐exchange columns, are discussed in the commentary.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R. 
Published: June 2004

AbstractReliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented below. These three procedures cover a range from 5 to 10 ng/ml DNA to 50 mg/ml DNA.
Palladium‐Mediated C5 Substitution of Pyrimidine Nucleosides
Authors: Ahmadian Mohammad , Klewer Douglas A. , Bergstrom Donald E. 
Published: June 2004

AbstractOne of the most efficient ways to link a reporter group to oligonucleotides is through the incorporation of a modified nucleoside during automated oligonucleotide synthesis. To be useful, it is important that the reporter group not interfere in hybridization reactions. This unit describes two linkers that can be used for the incorporation of a reporter group at the C5 position of deoxyuridine: a flexible aminoethylthioether linker, and a rigid amidopropynyl linker. The latter is suffciently long and positioned so that the reporter group lies outside the major groove of the DNA duplex.
Enzymatic Synthesis of M1G‐Deoxyribose
Authors: Schnetz‐Boutaud Nathalie C. , Chapeau Marie‐Christine , Marnett Lawrence J. 
Published: June 2004

AbstractAdducts formed between electrophiles and nucleic acid bases are believed to play a key role in chemically induced mutations and cancer. M1G‐dR is an endogenous exocyclic DNA adduct formed by the reaction of the dicarbonyl compound malondialdehyde with a dG residue in DNA. It is an intermediate in the synthesis of a class of modified oligodeoxyribonucleotides that are used to study the mutagenicity and repair of M1G. This unit presents methods for synthesizing M1G‐dR by enzymatic coupling.
Unnatural Nucleosides with Unusual Base Pairing Properties
Authors: Bergstrom Donald E. 
Published: June 2004

AbstractSynthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in nucleic acid biochemistry. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered including base pairs with alternative H‐bonding schemes, hydrophobic base pairs, degenerate bases, universal nucleosides, and triplex constituents.
Deoxyribonucleoside Phosphoramidites
Authors: Wilk Andrzej , Grajkowski Andrzej , Chmielewski Marcin K. , Beaucage Serge L. , Phillips Lawrence R. 
Published: June 2004

AbstractThe detailed preparation of deoxyribonucleoside phosphoramidites bearing a 4‐[N‐methyl‐N‐(2,2,2‐trifluoroacetyl)amino]butyl group for P(III) protection is presented. The use of this group circumvents nucleobase alkylation during oligonucleotide deprotection. Two syntheses of phosphoramidites starting from either a phosphordichloridite precursor or a bis‐(N,N‐diisopropylamino)chlorophosphine intermediate are described for the phosphinylation of suitably protected deoxyribonucleosides.
Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H‐Phosphonate Method
Authors: Strömberg Roger , Stawinski Jecek 
Published: June 2004

AbstractOligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.
Oligoribonucleotides with 2′‐O‐(tert‐Butyldimethylsilyl) Groups
Authors: Bellon Laurent 
Published: June 2004

AbstractThe chemical synthesis of oligoribonucleotides on solid support is routinely performed via the phosphoramidite method. However, the additional 2‐OH function of the ribofuranosyl sugar requires suitable protection during oligoribonucleotide synthesis. This unit describes methods for 2‐OH protection using the TBDMS group.