bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
Authors: Kahana Jason A. , Silver Pam A.
Published: June 2004

Fluorescent molecules serve as valuable tools for the detection of numerous biochemical phenomena and have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting and determination of chemical concentrations. However, the use of such techniques generally requires significant nonphysiological perturbations to the biological system being studied; therefore, they are not always appropriate for the observation of dynamic phenomena. Green fluorescent protein (GFP), cloned from jellyfish, has been used to overcome many of these problems. It is a small, extremely stable fluorescent protein that has been successfully expressed and detected in a wide variety of organisms, both in intact form and fused to other proteins. This overview unit describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena.
Green Fluorescent Protein in the Study of Neuronal Signaling Pathways
Authors: Blair Leslie , Bence‐Hanulec Kendra , Marshall John
Published: June 2004

In recent years, techniques have been established for transiently co‐transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non‐toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co‐transfecting with separate GFP and gene‐of‐interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene‐of‐interest and GFP cassettes. Also included is a method for the rapid, large‐scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.
Analysis ofRNAby Northern and Slot‐Blot Hybridization
Authors: Brown Terry , Mackey Karol
Published: June 2004

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re‐hybridized with a different probe.
Principles of Signal Conditioning
Authors: Finkel Alan , Bookman Richard
Published: June 2004

It is rare for biological, physiological, chemical, electrical, or physical signals to be measured in the appropriate format for recording and interpretation. Usually, a signal must be conditioned to optimize it for both of these functions. This overview describes the fundamentals of signal filtering, how to prepare signals for A/D conversion, signal averaging to increase the signal‐to‐noise ratio, line frequency pickup (hum), peak‐to‐peak and rms noise measurements, blanking, audio monitoring, testing of electrodes and the common‐mode rejection ratio.
Fabrication of Patch Pipets
Authors: Rae J.L. , Levis R.A.
Published: June 2004

Patch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole‐cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single‐channel and whole‐cell current measurements dictate how the pipets must be fabricated.
Acute Isolation of Neurons from the Mature Mammalian Central Nervous System
Authors: Kay Alan R. , Krupa David J.
Published: June 2004

The acute dissociation procedure provides a simple means of isolating neurons from the mature mammalian central nervous system. The method was primarily devised to isolate neurons for patch‐clamp electrophysiology. It may also prove useful for single‐cell PCR, immunocytochemistry, sorting of fluorescently labeled cells, or long‐term tissue culture of mature neurons. Dissociation is brought about by a combination of proteolysis and an ionic environment that encourages breakdown of the tissue. The method allows the isolation of neurons free of glial ensheathments in as little as 45 min after the sacrifice of the animal. Neurons so isolated lose fine dendritic branches, although the structure proximal to the cell body is often maintained, allowing identification of the morphological type of the neuron. The preparation has the following advantages: (1) the neurons are fully differentiated; (2) the cells are electronically compact, which improves the fidelity of the voltage clamp; (3) the cells are removed from the influence of surrounding cells; and (4) neurons can be isolated from small, circumscribed loci within the adult central nervous system.
Single‐Channel Recording
Authors: Jackson Meyer B.
Published: June 2004

This unit provides detailed descriptions for the steps of patch excision, data acquisition, and data analysis, and elaborates upon the relevant issues discussed in other units from Chapter 3. It includes discussions of the instrumentation for single‐channel recording and the key concepts necessary for the interpretation of single‐channel data.
Chronic Recording of Extracellular Neuronal Activity in Behaving Animals
Authors: Szymusiak Ronald , Nitz Douglas
Published: June 2004

Two methods for recording extracellular neuronal activity in unanesthetized, unrestrained rats are described in this unit. Both use chronically‐implanted bundles of fine microwires to record electrophysiological activity. One method provides recordings of single and/or multiple unit activity from individual wires in a bundle (monotrode). Discrimination of individual neuronal potentials is based on action potential amplitude, or on a combination of action potential amplitude and shape. The second method uses a 2‐ to 4‐microwire array (stereotrode‐tetrode) to yield multiple unit recordings. Discrimination of individual neuronal potentials is based on action potential shapes and the relative amplitude of action potentials recorded simultaneously on the different wires in the array. These methods can provide stable, long‐term recording of neuronal activity during a variety of behavioral paradigms.
Recording in the Cerebellar Slice
Authors: Swanson Geoffrey T. , Contractor Anis
Published: June 2004

This unit describes techniques for the preparation of mouse cerebellar slices and electrophysiological recording from neurons in the slice. The cerebellum provides a model of motor learning that can be correlated with alterations in synaptic function. In addition, the architecture and well‐defined synaptic pathways in the cerebellar slice make this preparation a useful model for exploring general principles of synaptic transmission.
Microdialysis in Nonhuman Primates
Authors: Saunders Richard C. , Kolachana Bhaskar S. , Weinberger Daniel R.
Published: June 2004

This unit presents a technique that allows for routine repeated microdialysis experiments in the monkey using a sedated preparation or, with further minor modification, in the awake behaving animal. Protocols are provided for construction and in vitro calibration of dialysis probes, obtaining magnetic resonance imaging (MRI) scans of the monkey brain, surgically attaching a guide holder to the skull, and performing postoperative MRI scanning, in vivo dialysis sample collection, anatomical verification of the probes, and neurochemical analysis. An alternate protocol describes microdialysis in awake, behaving monkeys; this requires substantial preparatory work in training the monkeys to sit quietly in a restraining chair and/or to perform a series of behavioral tasks.
Detection and Quantification of Neurotransmitters in Dialysates
Authors: Pani (catecholamines/indoleamines and acetylcholine) A.K. , Shippenberg (catecholamines/indoleamines) Toni S. , Heidbreder (acetylcholine) Christian , Espey (amino acids) Michael Graham
Published: June 2004

This unit describes methods for the separation and quantification of neurotransmitters, peptides, and second messengers in microdialysis samples by high‐pressure liquid chromatography (HPLC) coupled to either electrochemical detection (EC) or fluorescence detection. The protocols describe commonly used procedures for the detection of catecholamines and indoleamines (HPLC‐EC detection; detection of acetylcholine (HPLC‐EC detection with enzyme reactor), and detection of amino acids (HPLC‐fluorescence detection). Each of these protocols affords detection limits in the low nanomolar range.
Saturation Assays of Radioligand Binding to Receptors and Their Allosteric Modulatory Sites
Authors: Basile Anthony
Published: June 2004

The protocols in this unit describe methods for measuring radioligand binding to sites on the GABA‐A receptor complex. This system was chosen because of the fundamental role of GABA‐A receptors in central nervous system function and the presence of multiple binding sites on one supramolecular complex. However, these basic techniques can be used to analyze ligand binding to a wide variety of intracellular and extracellular sites associated with ion channels, transporters, or G proteins with relatively simple modifications of the protocols (e.g., tissue preparation, buffers, assay termination techniques, incubation times, and temperatures). A saturation assay is presented to examine ligand binding to the benzodiazepine site on the GABA‐A receptor, while a second protocol uses a similar technique to examine the binding site for GABA in this complex.
Measurement of Chloride Movement in Neuronal Preparations
Authors: Schwartz‐Bloom Rochelle D. , Engblom A. Christine , Åkerman Karl E. O. , Inglefield Jon R.
Published: June 2004

In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand‐gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma‐aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride‐sensitive fluorescent dyes.
Measurement of Cation Movement in Primary Cultures Using Fluorescent Dyes
Authors: Reynolds Ian J.
Published: June 2004

Ca2+, Na+, K+, and Mg2+ have a central role in neuronal excitability. The concentration of these cations in the cytoplasm of neurons (generically termed [ion+]i) provides a marker of the excitation state of the neurons, and may also illuminate the activity of specific signaling mechanisms that involve Ca2+‐ or Mg2+‐activated enzymes. The measurement of [ion+]i in cultured neurons is achieved with the use of an ion‐sensitive fluorescent dye in combination with equipment designed to quantitatively measure fluorescence. Specificity is obtained by choosing dyes with the appropriate selectivity for the ion of interest. Measurements of steady state ion concentrations can be made, as well as measurements of the net difference between ion movement into the cytoplasm (in response to a stimulus) and the physiological buffering of that ion. The procedures in this unit for loading and recording from dyes are broadly similar for each ion when ratiometric dyes are used as described, and can readily be modified for use with single‐wavelength dyes. Support protocols are provided for calibration of individual dyes, which can be more problematic.
Scintillation Proximity Assay
Authors: Kahl Steven D. , Felder Christian C.
Published: June 2004

Scintillation proximity assay technologies provide a rapid non‐separation method to measure common biological interactions. This unit identifies potential uses of the technology for the measurement of receptor‐ligand binding, cAMP accumulation, protease activity and cellular uptake.
In Vivo Measurement of Blood‐Brain Barrier Permeability
Authors: Boje Kathleen M.K.
Published: June 2004

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood ‐ brain barrier. Methods for the measurement of drug influx or efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier‐mediated or saturable transport.
Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes in Tissue Homogenates: ELISA Assay of Amyloid β‐Peptides
Authors: Hyslop Paul A. , Bender Mark H.
Published: June 2004

Use of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.
Sexual and Reproductive Behaviors
Authors: Sisk Cheryl L. , Meek Leslie R.
Published: June 2004

The procedures described in this unit include testing procedures for male and female reproductive behaviors, gonadectomy, and hormonal treatments appropriate for inducing male and female reproductive behaviors. Because reproductive behaviors are social behaviors, and therefore require the presence of stimulus animals, the protocols in this unit also provide information on the preparation of stimulus animals. The protocols are written for use with laboratory rats, although a discussion of issues related to species differences in the study of reproductive behaviors is included.
Application of Experimental Stressors in Laboratory Rodents
Authors: Heinrichs Stephen C. , Koob George F.
Published: June 2004

This unit presents seven separate stressor protocols for laboratory rodents. Stress induction is a critical element in the study of neural and neuroendocrine mechanisms involved in establishing and maintaining a state of stress. The first three procedures, immobilization, footshock, and swimming, involve acute exposure to noxious stimuli. The next three procedures, social isolation, resident/intruder aggression, and maternal deprivation, induce social disruption by withdrawal from a group housing condition, attack within the unfamiliar territory of a dominant male, or segregation of a preweanling pup from its mother, respectively. The final procedure, sleep deprivation, involves passive denial of the opportunity to sleep. Support protocols are provided to address the need for environmental acclimation and calming procedures prior to any stress‐related studies (including, for rats, handling of the animals as a calming measure) and to detail a simple method of quantifying the response to a given stressor by direct measurement of levels of the stress hormones adrenocorticotrophic hormone (ACTH) and corticosterone.
Cued and Contextual Fear Conditioning in Mice
Authors: Wehner Jeanne M. , Radcliffe Richard A.
Published: June 2004

Contextual and cued fear conditioning is a robust form of learning in which an association is made between stimuli and their aversive consequences. Fear conditioning has been used in laboratory rodents in part because it is a highly conserved form of behavior that is exhibited in both laboratory situations and in normal environments. Training requires only a single trial and this makes it adaptable to genetic, pharmacological, and biochemical studies. Clinically, it is has relevance to human behavior in that fear conditioning can be produced in humans, and damage to the amygdala prevents fear conditioning.
Assessment of Sustained and Divided Attention in Rats
Authors: Arnold H. Moore , Bruno John P. , Sarter Martin
Published: June 2004

Behavioral tasks must be evaluated in terms of the cognitive functions they require in order to be performed. All of the tasks described in this chapter can be used with each of four experimental manipulations: stimulation of a single brain region by drugs or small electrical current, impairment of normal function by production of a lesion or administration of appropriate pharmacological agents, recording of brain activity during the performance of a specific behavioral task, or behavioral phenotyping of transgenic and knockout mice for genes expressed in specific brain regions. This unit describes protocols for the radial arm maze task and the water maze task, both of which require intact spatial memory abilities.
Overview of Methodological Approaches to the Study of Ingestive Behavior
Authors: Hoebel Bartley G. , Wellman Paul J.
Published: June 2004

Some examples of procedures used by feeding researchers are discussed in this unit and include ablation of neural function; inhibition of behaviors by selective neurotoxins and antisense oligonucleotides; staining of sensory and motor mechanisms; electrical stimulation of the brain; local injection and microdialysis of nutrients, neurotransmitters, and drugs; autoradiography and in situ hybridization of neurotransmitters with their receptors; electrophysiological techniques for multi‐ and single‐unit recording of cells in the hypothalamus; and gene technology using inbred strains of genetically obese mice.
Basic Measures of Food Intake
Authors: Wellman Paul J. , McMahon Lance R.
Published: June 2004

This unit describes a method for measuring the consumption of a pellet diet during a 30‐min testing session conducted during the late portion of the day. The procedure generates reliable and stable baseline measures of feeding, and is appropriate for assessing experimental manipulations that either enhance (e.g., injecting a peptide into the brain) or suppress feeding (e.g., systemic injections of amphetamine).
Direct, Continuous Behavioral Analysis of Drug Action on Feeding
Authors: Halford Jason , Blundell John
Published: June 2004

Analysis of rodent feeding behavior during the study of anorectic drugs reveals that contextual variables such as type of food, drug, environment, and physiological state influence food intake. Temporal changes in behavior can signal the onset of a feeding modulation process, as is the case with anorectic drugs acting on distinct neurochemical mechanisms that change temporal behavioral profiles. Comparing the effects of anorectic agents in controlled animal assays can identify potential therapeutic drugs that may influence human eating behavior. This unit specifies the materials and procedures required for continuously monitoring the Behavioral Satiety Sequence, as well as a device for continuous, automatic measurement of the mass of food consumption by the animal during behavioral monitoring called the RUEMA (Rat Universal Eating Monitor Apparatus).
Conditioned Flavor Preferences: Evaluating Postingestive Reinforcement by Nutrients
Authors: Ackroff Karen , Sclafani Anthony
Published: June 2004

The acceptance of food and the expression of preferences for particular foods have both innate and learned components. To determine the mechanism(s) responsible for the acquisition of learned preferences and the enhancement of inborn taste preferences, it is important to separate the component stimuli: the oral flavor cues and the postingestive nutrient cues that are associated with the flavors. Unambiguous analysis of postingestive reinforcement of a nutrient requires post‐oral presentation of the nutrient during or after oral intake of the flavor stimulus. This protocol concentrates on controlling stimulus presentation in associative conditioning, pairing a flavor with positive postingestive effects.
Measurement of Rodent Stereotyped Behavior
Authors: Kelley Ann E.
Published: June 2004

This unit presents a quantitative, observational method for the assessment of rodent stereotyped behavior which consists of motor responses that are repetitive, invariant, and seemingly without purpose or goal. The most classic behavioral pattern that is characteristic of stereotypy is that elicited by high doses of stimulants, such as cocaine and amphetamine, in rats, although it can also occur in response to other drugs or neurotoxic treatments affecting the basal ganglia. An observational time‐sampling procedure is described in which animals are observed and rated by an experimenter, who is blind to treatment, at regular time points over the course of a behavioral testing period. The frequency of different behaviors is measured by scoring the presence or absence of a given behavior during predetermined time bins. The apparatus and test procedures are described, and a comprehensive list of commonly observed behaviors that may appear as stereotyped is provided. In addition to being ideally suited to the measurement of stereotypy, the protocol can be adapted to sampling many forms of spontaneous behaviors, including locomotion, rearing, grooming, eating, and drinking. Samples of behavioral checklists and scoring sheets are also provided.
Motor Coordination and Balance in Rodents
Authors: Carter Rebecca J. , Morton Jennifer , Dunnett Stephen B.
Published: June 2004

Measurement of motor coordination and balance can be used not only to assess the effect of drugs or other experimental manipulations on mice and rats, but also to characterize the motor phenotype of transgenic or knock‐out animals. Three well established and widely used protocols for measuring motor coordination and balance in mice and rats (rotarod, beam walking and footprint analysis) are described in this unit. The tests can be used equally well for rats and mice, and have been used both for the phenotypic characterization of transgenic mice and for evaluating the effects of lesions and aging in rats. The protocols are described in the primary context of testing mice, but modifications of the test apparatus or variations in the test parameters for assessment of rats are noted.
Parental Behaviors in Rats and Mice
Authors: Lonstein Joseph S. , Fleming Alison S.
Published: June 2004

Parental care in mammals is often critical for survival and development of the offspring. The multiple behaviors that comprise parental care in rats and mice are controlled by a complex array of factors. This unit describes the components of parental care in rats and mice and the frequently used methods to assess the performance of these behaviors, a subject's preference for pup‐related cues, as well as a subject's motivation to act parentally.
Assessment of Murine Startle Reactivity, Prepulse Inhibition, and Habituation
Authors: Geyer Mark A. , Dulawa Stephanie C.
Published: June 2004

The study of the startle response and its plasticity provides a powerful research paradigm for examining the neural control of behavior. The neural substrates underlying phenomena such as information‐processing functions, including sensorimotor gating, affective modulation, and habituation, can be studied using startle plasticity as an operational measure. The mouse is rapidly becoming the most powerful model system for the study of human CNS function and is highly amenable to both genetic and pharmacological study. This unit describes procedures for the assessment of the startle response to acoustic stimuli in mice; methods for assessing prepulse inhibition (PPI) of startle, with treatments including drug administration and genetic manipulation; and methods for testing habituation.
Dural Inflammation Model of Migraine Pain
Authors: Phebus Lee A. , Johnson Kirk W.
Published: June 2004

The cause of migraine pain is controversial. One recently proposed theory is that migraine pain may originate from inflammation of the meninges, particularly the dural membranes that surround the brain. This theory proposes that, during a migraine, there is an idiopathic activation of trigeminal sensory afferents, resulting in nociceptive transmission to the CNS as well as the release of pro‐inflammatory substances in the periphery, particularly the dura. Dural inflammation is thought to lower the nociceptive threshold of dural afferents and facilitate nociceptive transmission to the central nervous system. In the procedure described in this unit, trigeminal sensory afferents are activated by electrically stimulating the trigeminal ganglion. This stimulation causes trigeminal peripheral sensory afferents to depolarize, inflammatory substances to be released from these afferents, and dural inflammation to appear. Dural inflammation is quantified by measuring plasma protein extravasation. The basic protocol describes this model in rats, and the alternate protocol describes the analogous procedure in guinea pigs.
Rodent Models of Focal Cerebral Ischemia
Authors: O'Neill Michael J. , Clemens James A.
Published: June 2004

This unit presents models that are both used to study ischemic mechanisms and to test for neuroprotective agents or agents that enhance recovery from stroke. The Tamura model is one of the best characterized focal ischemia models in which the middle cerebral artery is occluded by electrocoagulation. Also described is the intraluminal monofilament model, the spontaneously hypertensive rat (SHR), and the newer endothelin‐1 model. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.
A Murine Model of Retroviral Encephalopathy
Authors: Sei Yoshitatsu , Basile Anthony S.
Published: June 2004

This unit delineates the steps for production of a murine model of retroviral encephalopathy. The LP‐BM5 infected mouse develops a chronic inflammation of the brain secondary to profound immune deficiency. The model is robust, develops rapidly and does not require the use of human pathogens. In addition, the behavioral and neurochemical characteristics of this model is reviewed.
Intravenous Self‐Administration of Ethanol in Mice
Authors: Grahame Nicholas J. , Cunningham Christopher L.
Published: June 2004

A more complete understanding of alcohols reinforcing actions is obtained when multiple behavioral procedures are used, some of which bypass taste factors. This unit describes a method for assessing the reinforcing effects of alcohol in mice using the most widely accepted procedure for assessing drug reward: intravenous self‐administration.
Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in Treating Alcoholism and Studying the Neuropharmacological Bases of Ethanol‐Seeking Behaviors in Rats
Authors: June Harry L.
Published: June 2004

The unit outlines four basic protocols designed to systematically evaluate the capacity of potential pharmacotherapeutic agents to effectively treat alcohol addiction and dependence in rats. Also included are procedures designed to study the neural mechanisms regulating alcohol‐seeking behaviors.
Measurement of Panic‐Like Responses Following Intravenous Infusion of Sodium Lactate in Panic‐Prone Rats
Authors: Sajdyk Tammy J. , Keim Stanley R. , Thielen Shelley R. , Fitz Stephanie D. , Shekhar Anantha
Published: June 2004

This unit describes a putative animal model for panic disorder. The basic premise is that pharmacological disruption of critical brain regions implicated in the circuitry of anxiety will lead to a condition similar to that of the human disorder. A clinically relevant test, the sodium lactate challenge, is utilized to assess parallels between the human condition and this rat model.
Patch‐Pipet Recording in Brain Slices
Authors: Stuart Greg
Published: June 2004

Patch‐clamp recording in brain slices provides a powerful approach for investigating the intrinsic electrical properties of neurons and glia and analyzing synaptic interactions between neurons. There are two main methods presently in use to obtain patch‐clamp recordings from neuronal or glial cell bodies and their processes in brain slices, both described in this unit. In the blind technique described in this unit, the approach to a cell is performed blindly and recordings are obtained relying purely on electrical cues. Alternatively, the movement of the recording pipet through the brain slice and its placement onto the cell membrane can be performed as described under visual control; this method is usually performed with the aid of differential interference contrast (DIC) optics and is referred to as the DIC technique. Increased resolution of cells and their processes can be obtained by combining DIC optics with infrared illumination and video microscopy techniques. Using either of these methods it is possible to reliably apply the full power of the patch‐clamp technique to the study of neurons and glial cells in brain slices.
Optimization of Transfection
Authors: Rose John K.
Published: June 2004

The single most important factor in optimizing transfection efficiency is selecting the proper transfection protocol. This overview discusses the most commonly used procedures: calcium phosphate‐mediated gene transfer, DEAE‐dextran‐mediated gene transfer, electroporation, and liposome‐mediated transfection. Fusion techniques such as protoplast fusion and microinjection may also be considered.
Preparation of RNA from Tissues and Cells
Authors: Ribaudo Randall , Gilman Michael , Kingston Robert E. , Chomczynski Piotr , Sacchi Nicoletta
Published: June 2004

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA. The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

The first section of this unit discusses basic principles of sterile technique, while the second section discusses medium preparation. The basic protocol describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Syntheses of Specifically 15N‐Labeled Adenosine and Guanosine
Authors: Gaffney Barbara L. , Jones Roger A.
Published: June 2004

This unit describes the specific incorporation of 15N into the N7 and amino positions of adenosine, and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions. Two variations of the procedures are also presented that include either 12C or 13C at the C8 position of adenosine, and 13C at either the C8 or C2 position of guanosine. These 13C tags permit the incorporation of two 15N‐labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C‐N coupling. While the major application of these specifically 15N‐labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. A support protocol describes the synthesis of 7‐methylguanosine.
Synthesis of Protected 2′‐Deoxy‐2′‐fluoro‐β‐D‐arabinonucleosides
Authors: Elzagheid Mohamed I. , Viazovkina Ekaterina , Damha Masad J.
Published: June 2004

This unit describes in detail the preparation of protected 2'‐deoxy‐2'‐fluoroarabinonucleosides. These building blocks are required for the synthesis of 2'‐deoxy‐2'‐fluoroarabinonucleic acid (2'F‐ANA), an oligonucleotide analog exhibiting very promising antisense properties. The preparation of phosphoramidites from these building blocks and the synthesis of 2'F‐ANA are described elsewhere in the manual.
Synthesis of 1,5‐Anhydrohexitol Building Blocks for Oligonucleotide Synthesis
Authors: Lagoja Irene M. , Marchand Arnaud , Van Aerschot Arthur , Herdewijn Piet
Published: June 2004

This unit describes in detail, the optimized preparations of 1,5‐anhydrohexitol and the 1,5‐anhydrohexitol building blocks for oligonucleotide synthesis (hG, hA, hC, hT).
Protection of 5′‐Hydroxy Functions of Nucleosides
Authors: Seliger H.
Published: June 2004

The 5‐hydroxy group is the primary hydroxy group of nucleosides. It is mandatory to protect 5‐hydroxyls in all methods of oligonucleotide synthesis that require nucleoside synthons. This unit discusses a wide variety of acid‐labile and base‐labile protecting groups, as well as enzymatic methods for 5‐protection and deprotection.
Deoxyribo‐ and Ribonucleoside H‐Phosphonates
Authors: Stawinski Jacek , Strömberg Roger
Published: June 2004

Most methods for preparing H‐phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation prodecures that it compatible with most common protecting groups.
Regioselective 2′‐Silylation of Purine Ribonucleosides for Phosphoramidite RNA Synthesis
Authors: Gaffney Barbara L. , Jones Roger A.
Published: June 2004

This unit describes high‐yield procedures for protection of purine ribonucleosides based on a reaction that allows concomitant highly regioselective 2′‐silylation and 3′‐phosphitylation to give, in one step, monomers that are ready for H‐phosphonate synthesis. For preparation of phosphoramidites, the H‐phosphonate monoester is cleaved without silyl migration to give intermediates ready for phosphitylation by standard methods.
Preparation of 2′‐O‐[(Triisopropylsilyl)oxy]methyl‐protected Ribonucleosides
Authors: Pitsch Stefan , Weiss Patrick A.
Published: June 2004

The [(triisopropylsilyl)oxy]methyl (TOM) group is a useful protecting group for the 2′‐OH of ribonucleosides to be used for oligoribonucleotide synthesis by the phosphoramidite method. It is completely stable to all reaction conditions applied during assembly and the first deprotection step. It does not interfere with the coupling reaction and leads to very good coupling yields under DNA‐coupling conditions. The final cleavage occurs quantitatively without concomitant destruction of the RNA product. This unit describes the synthesis and characterization of 2′‐O‐TOM‐5′‐O‐dimethoxytrityl‐N‐acetyl ribonucleosides in full detail. The TOM‐group is introduced via a dibutyltin dichloride‐mediated reaction into N‐acetylated, 5′‐O‐dimethoxytritylated ribonucleosides. Support protocols describe the synthesis of N‐acetylated, 5′‐O‐dimethoxytritylated adenosine and guanosine, as well as synthesis of the starting reagent [(triisopropylsilyl)oxy]methyl chloride (TOM‐Cl). Preparation of the phosphoramidites and their use in solid‐phase oligonucleotide synthesis are described elsewhere in the series.
Attachment of Nucleosides to Solid‐Phase Supports
Authors: Pon Richard T.
Published: June 2004

Specific step‐by‐step instructions are given for coupling nucleosides to LCAA‐CPG supports (supports consisting of a long‐chain alkylamine linked to controlled‐pore glass). Protocols are given for a succinic acid linker and a hydroquinone‐O,O′‐diacetic acid linker. The former is the most widely used linker arm, and the starting materials are widely available. The latter offers greater compatibility with base‐sensitive sequence modifications and great synthetic throughput because it can be cleaved under milder and faster conditions. Additional guidelines are given for selecting a linker arm and coupling protocol. Almost any application requiring synthetic oligonucletodies can be satisfied using one of these linker arms.
Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
Authors: Beaucage Serge L. , Caruthers Marvin H.
Published: June 2004

The phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.
Strategies for Oligoribonucleotide Synthesis According to the Phosphoramidite Method
Authors: Wincott Francine E.
Published: June 2004

Advances in oligoribonucleotide synthesis have lagged behind those in oligodeoxyribonucleotide synthesis because of the difficulty in identifying orthogonal protecting groups for the 2′‐ and 5′‐hydroxyls. Adaptation of the phosphoramidite method for DNA synthesis to RNA synthesis has greatly improved our understanding of RNA. It allows site‐specific introduction of modified nucleosides to any position in an RNA molecule, as well as introduction of variations at multiple sites in the molecule. This overview discusses issues that are relevant to RNA synthesis by the phosphoramidite approach, including supports used, activation of the ribonucleoside phosphoramidites, and protection of the nucleobase, phosphate, and 2′‐ and 5′‐hydroxyls.
Direct Attachment of Conjugate Groups to the 5′ Terminus of Oligodeoxyribonucleotides
Authors: Asseline Ulysse , Thuong Nguyen T.
Published: June 2004

This unit gives protocols for the attachment of intercalating and photoreactive conjugate groups to oligodeoxyribonucleotides. Protocols are given for acridine‐ and psoralen‐conjugated oligonucleotides, and include attachment of the linker, preparation of the phosphoramidite, coupling to the oligonucleotide, deprotection, purification, and characterization.
Attachment of Reporter and Conjugate Groups to the 3′ Termini of Oligonucleotides
Authors: Greenberg Marc M.
Published: June 2004

Conjugation of oligonucleotides at the 3 terminus is less common because this site is used for covalent linkage to solid‐phase oligonucleotide synthesis supports. However, 3‐oligonucleotide conjugates have several valuable physicochemical properties, including their ability to stabilize nucleic acid hybridization complexes and to retard the activity of exonucleases. This unit discusses methods for preparing oligonucleotides conjugated at the 3 terminus.
Incorporation of Halogenoalkyl, 2‐Pyridyldithioalkyl, or Isothiocyanate Linkers into Ligands
Authors: Asseline Ulysee , Thuong Nguyen T.
Published: June 2004

Ligands can be introduced at the 5' terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5' terminus of the oligonucleotide. These are then reacted to give the ligand‐oligonucleotide conjugate. The addition of appropriate linkers to ligands is described in this unit. 5'Modification of the oligonucleotide and the final reaction that produces the ligand‐conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite‐ or H‐phosphonate‐mediated oligonucleotide synthesis.
Conjugation of 5′‐Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands
Authors: Asseline Ulysse , Thuong Nguyen T.
Published: June 2004

This unit reports the synthesis of oligodeoxyribonucleotides covalently linked via their 5′ termini to various ligands such as intercalating agents, reactive groups, or labels. Methods for incorporation of halogenoalkyl, isothiocyanate, and 2‐pyridyldisulfide linkers onto ligands, and addition of amino, carboxyl, thiophosphate, phosphate, and masked thiol groups at the 5 terminus of an oligodeoxyribonucleotide are described elsewhere in the series. This unit reports procedures for coupling the ligands and oligonucleotides, as well as details for purification and characterization of the final products.
Solid‐Phase Synthesis of Branched Oligonucleotides
Authors: Carriero Sandra , Damha Masad J.
Published: June 2004

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.
Chemistry of CpG DNA
Authors: Kandimalla Ekambar R. , Agrawal Sudhir
Published: June 2004

The vertebrate immune system can recognize specific pathogen‐associated molecular patterns in invading microorganisms, including the unmethylated CpG dinucleotide. This unit discusses the receptors that recognize CpG motifs and important aspects of the sequence context of CpG motifs to the end of understanding and designing CpG DNA for therapeutic purposes.
Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages
Authors: Guga Piotr , Stec Wojciech J.
Published: June 2004

A method for solid‐phase synthesis of stereodefined PS‐oligos via an oxathiaphospholane approach using pure P‐diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5′‐O‐DMTr‐N‐protected deoxyribonucleosides with 2‐chloro‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O‐labeled mercaptoalcohol, the corresponding 18O‐labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS‐oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s into their P‐diastereomers, and for manual solid‐phase synthesis of PS‐oligos. Oxidation of 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s with selenium dioxide yields their 2‐oxo‐analogs, which are suitable either for elongation of stereodefined PS‐oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O‐labeled PO‐oligos of predetermined P‐chirality.
Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification
Authors: Fang Shiyue , Bergstrom Donald E.
Published: June 2004

The preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5′‐terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5′‐OH and 5′‐phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin‐coated microspheres, full‐length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high‐quality, full‐length, unmodified 5′‐OH and 5′‐phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.
Stepwise Solid‐Phase Synthesis of Nucleopeptides
Authors: Grandas Anna , Marchán Vicente , Debéthune Laurent , Pedroso Enrique
Published: June 2004

Phosphodiester‐linked peptide‐oligonucleotide conjugates (nucleopeptides) are obtained by stepwise solid‐phase procedures. The peptide is first assembled on a suitably derivatized solid matrix and the oligonucleotide is subsequently elongated at the free hydroxyl group of the linking amino acid. Temporary acid‐labile and permanent base‐labile protecting groups are combined. Careful choice of the protection scheme is required to prevent and minimize side reactions that may degrade the target molecule.
Chemical and Enzymatic Methods for Preparing Circular Single‐Stranded DNAs
Authors: Diegelman Amy M. , Kool Eric T.
Published: June 2004

Small circular oligonucleotides can be used for diagnostic, therapeutic, and laboratory purposes. These systems have gained considerable attention in recent years because they form unusually strong and specific complexes with RNA and DNA strands. Synthetic circular DNAs of 20 to 200 nucleotides can also serve as catalysts for amplified DNA and RNA synthesis by a rolling circle mechanism. This unit presents methods for synthesizing small circular oligonucleotides. These simple “one‐pot” procedures are carried out using short DNA splints that hold the circle together until it is chemically or enzymatically ligated.