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Cell Line: Established cell cultures that have the potential to propagate indefinitely.
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Basic Chick Care and Maintenance

JoVE 5154

Chicks (Gallus gallus domesticus) are a valuable research tool, not only for studying important concepts in vertebrate development, neuroscience, and tumor biology, but also as an efficient system in which to propagate viruses. Although eggs can be purchased from external suppliers and working with chicks requires very little specialized equipment, an understanding of proper handling procedures is required for normal embryo development. This video will provide an overview of egg handling principles, including an explanation of the incubation parameters that can profoundly impact development: temperature, humidity, and egg rotation. Most experiments that use chicken eggs require access to the embryo within the shell, which is achieved by cutting a small, resealable hole, or “window.” This process is described in step-by-step detail, along with several other techniques essential for working with chicks, such as candling and India ink injection. Finally, the video will review some practical applications of these basic techniques in advanced scientific research.


 Biology II

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An Introduction to the Zebrafish: Danio rerio

JoVE 5128

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their high degree of genetic conservation with humans and their simple, inexpensive maintenance. Additionally, gene expression can be easily manipulated in zebrafish embryos, and their transparency allows for observation of developmental processes. This overview video first introduces basic zebrafish biology, including their phylogeny, life cycle, and natural environment, before presenting the features that make them so useful in the lab. A brief history of zebrafish research is also provided through a review of major discoveries made in fish, ranging from the early establishment of methods for efficient genetic screening to the discovery of novel therapeutics for human diseases such as cancer. Finally, some of the many avenues of experimentation performed in zebrafish are discussed, including immunological and developmental studies.


 Biology II

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Zebrafish Reproduction and Development

JoVE 5151

The zebrafish (Danio rerio) has become a popular model for studying genetics and developmental biology. The transparency of these animals at early developmental stages permits the direct visualization of tissue morphogenesis at the cellular level. Furthermore, zebrafish are amenable to genetic manipulation, allowing researchers to determine the effect of gene expression on the development of a vertebrate with a high degree of genetic similarity to humans. This video provides a brief overview of the major phases of zebrafish development, with particular focus on the first 24 hours post fertilization (hpf). The discussion begins with a zygote consisting of a single cell, or blastomere, atop a large ball of yolk. Cleavage of the blastomere is then shown to produce an embryo containing thousands of cells within a matter of hours. Next, the dramatic cellular movements known as epiboly and gastrulation are explained, revealing how they contribute to reshaping a mass of cells into a moving embryo with a beating heart in just 1 day. The presentation follows embryo development through the hatching phase, when they become swimming, feeding larvae. Important considerations for caring for larvae are incorporated, including a brief review of how fish are raised to adulthood in a dedicated facility known as the nursery. Finally, the video concludes with some commo


 Biology II

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Zebrafish Breeding and Embryo Handling

JoVE 5150

Zebrafish (Danio rerio) are an important model organism that is particularly valuable for research in developmental biology. Zebrafish are extremely fertile and can produce hundreds of progeny per week, so it is relatively easy to collect a large number of embryos for high sample numbers. Furthermore, zebrafish undergo rapid development and embryos are transparent, allowing for easy visualization of developmental processes. This video covers the steps required for the collection of newly fertilized zebrafish embryos. A brief overview of zebrafish mating behavior is presented, followed by instructions for setting up crosses in specialized laboratory breeding tanks that allow for controlled mating. Also covered are the conditions required to initiate the release of eggs (known as spawning) the morning after tanks are set. Next, essential techniques for working with embryos are presented, including the inhibition of pigment development with the chemical PTU, and dechorionation: a procedure in which the shell-like membrane surrounding the embryo (the chorion) is removed. Finally, the video concludes with some practical applications of these techniques in developmental research.


 Biology II

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Development of the Chick

JoVE 5155

The chicken embryo (Gallus gallus domesticus) provides an economical and accessible model for developmental biology research. Chicks develop rapidly and are amenable to genetic and physiological manipulations, allowing researchers to investigate developmental pathways down to the cell and molecular levels.

This video review of chick development begins by describing the process of egg fertilization and formation within the chicken reproductive tract. Next, the most commonly used chick staging nomenclature, the Hamburger Hamilton staging series, is introduced. Major events in chick development are then outlined, including the dramatic cellular movements known as gastrulation that form the three major cell layers: The ectoderm, mesoderm, and endoderm. Cells from these layers go on to generate all the tissues within the organism, as well as extraembryonic membranes, which are necessary for the transport of gases, nutrients, and wastes within the eggshell. To conclude the discussion, some exciting techniques will be presented as strategies for studying chick development in greater detail.


 Biology II

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Zebrafish Maintenance and Husbandry

JoVE 5152

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to their small size, large numbers of zebrafish can be housed in the laboratory at low cost. Although zebrafish are relatively easy to maintain, special consideration must be given to both diet and water quality to in order to optimize fish health and reproductive success. This video will provide an overview of zebrafish husbandry and maintenance in the lab. After a brief review of the natural zebrafish habitat, techniques essential to recreating this environment in the lab will be discussed, including key elements of fish facility water recirculation systems and the preparation of brine shrimp as part of the zebrafish diet. Additionally, the presentation will include information on how specific zebrafish strains are tracked in a laboratory setting, with specific reference to the collection of tail fin samples for DNA extraction and genotyping. Finally, experimental modifications of the zebrafish environment will be discussed as a means to further our understanding of these fish, and in turn, ourselves.


 Biology II

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An Introduction to the Chick: Gallus gallus domesticus

JoVE 5153

The chicken embryo (Gallus gallus domesticus) is an extremely valuable model organism for research in developmental biology, in part because most of their development takes place within an egg that is incubated outside of the mother. As a result, early developmental stages can be accessed, visualized and manipulated by simply creating a small hole in the eggshell. Since billions of chickens are raised worldwide for meat and egg production, scientists can easily and economically acquire large numbers of fertilized eggs throughout the year. Furthermore, chickens share significant genetic conservation with humans, so the genetic mechanisms that have been found to regulate chicken development are also relevant to our own biology. This video focuses on introducing the domesticated chicken as a scientific model. The discussion begins with a review of chicken phylogeny, revealing the features that make them amniotes, like other birds, reptiles, and mammals. Highlights from the millennia of chicken research will be presented, ranging from Aristotle’s postulates about the function of extra-embryonic membranes to more recent, Nobel-prize winning discoveries in neuroscience. Additionally, some current examples of studies performed in chicken embryos will be provided, such as in vivo tracking of cell movements during development and the recruitment of


 Biology II

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Chick ex ovo Culture

JoVE 5157

One strength of the chicken (Gallus gallus domesticus) as a model organism for developmental biology is that the embryo develops outside the female and is easily accessible for experimental manipulation. Many techniques allow scientists to examine chicken embryos inside the eggshell (in ovo), but embryonic access can be limited at later stages of development. Fortunately, chicks can also be cultured ex ovo, or outside of the eggshell. The major advantage to ex ovo culture is greater access to tissues that might otherwise be obstructed by the shell or the orientation of the chick within the egg, especially for embryos in later stages of development. There are two principle strategies to ex ovo culture: whole yolk culture and explant culture. During whole yolk culture, the eggshell is cracked and the contents are transferred to a simple housing vessel. However, in explant culture methods, the embryo is excised from the yolk and mounted in the housing vessel to maintain membrane tension, which is important for normal development. Basic protocols for whole-yolk and explant techniques will be provided in this video, along with a discussion of the pros and cons of culturing chicks outside of the shell. Finally, experimental applications of ex ovo culture will be discussed, demonstrating how this


 Biology II

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In ovo Electroporation of Chicken Embryos

JoVE 5156

Electroporation is a technique used in biomedical research that allows for the manipulation of gene expression via the delivery of foreign genetic material into cells. More specifically, in ovo electroporation is performed on early developing chicks (Gallus gallus domesticus) contained within their eggshells. In this procedure, DNA or knockdown constructs are first injected into a target tissue. However, the genetic material is unable to penetrate the plasma membrane to carry out its function within the cell. To solve this problem, an electrical field is applied, causing temporary disruptions to membrane stability. This electric field also causes the negatively charged nucleic acids to migrate toward the positively charged electrode through the holes in the plasma membrane, thus effectively driving the DNA or knockdown construct into the cell. The major advantage of this technique is that the delivery of genetic material can be localized to isolated cell types at specific developmental time points. As a result, the genetic mechanisms that govern individual developmental events can be examined. This video provides an overview of the principles behind in ovo electroporation and introduces the tools required for the technique, including capillary needles, electrodes, and an electroporator. A step-by-step protocol for carrying out the procedu


 Biology II

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An Introduction to Neurophysiology

JoVE 5201

Neurophysiology is broadly defined as the study of nervous system function. In this field, scientists investigate the central and peripheral nervous systems at the level of whole organs, cellular networks, single cells, or even subcellular compartments. A unifying feature of this wide-ranging discipline is an interest in the mechanisms that lead to the generation and propagation of electrical impulses within and between neurons. This subject is important not only for our understanding of the fascinating processes driving human thought, but also for our ability to diagnose and treat disorders related to nervous system malfunction. This video will provide an introduction to the field of neurophysiology, beginning with a brief history of neurophysiological research that showcases landmark studies like Galvani’s observations of twitching frog legs and Eccles’s discovery of the chemical synapse. Next, key questions asked by neurophysiologists are introduced, followed by an overview of some prominent experimental tools used to answer those questions. The methods presented range from techniques used to investigate single cells, like patch clamping, to those that can measure activity across large regions of the brain, like electroencephalography (EEG). Finally, applications of neurophysiological research are discuss


 Neuroscience

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An Introduction to Neuroanatomy

JoVE 5204

Neuroanatomy is the study of nervous system structures and how they relate to function. One focus of neuroanatomists is the macroscopic structures within the central and peripheral nervous systems, like the cortical folds on the surface of the brain. However, scientists in this field are also interested in the microscopic relationships between neurons and glia - the two major cell types of the nervous system. This video provides a brief overview of the history of neuroanatomical research, which dates back to the 4th century BC, when philosophers first proposed that the soul resides in the brain rather than the heart. Key questions asked by neuroanatomists are also reviewed, including topics like the role cytoarchitecture, or the arrangement of neurons and glia, plays in brain function; and how neuroanatomy changes as a result of experience or disease. Next, some of the tools available to answer these questions, such as histology and magnetic resonance imaging, are described. Finally, the video provides several applications of neuroanatomical research, demonstrating how the field lives on in today’s neuroscience labs.


 Neuroscience

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An Introduction to Developmental Neurobiology

JoVE 5207

Developmental neuroscience is a field that explores how the nervous system is formed, from early embryonic stages through adulthood. Although it is known that neural progenitor cells follow predictable stages of proliferation, differentiation, migration, and maturation, the mechanisms controlling the progression through each stage are incompletely understood. Studying development is not only important for understanding how complex structures are assembled, but also for characterizing and treating developmental disorders. Since injury repair processes are similar to those that occur in development, this field is also a promising source of insight into when and how nervous system tissues regenerate.This video provides a brief overview of the field of developmental neuroscience, including some key experiments that have advanced our understanding of the mechanisms controlling the formation of early neural tissue and the further specialization of those cells into discrete subsets of neurons. The discussion focuses on prominent questions that developmental biologists are asking and then demonstrates some of the methods that they use to investigate these questions. Finally, applications of the techniques are presented to provide insight into what it means to be a developmental neuroscientist today. The range of experiments demo


 Neuroscience

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Murine In Utero Electroporation

JoVE 5208

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.


 Neuroscience

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Explant Culture of Neural Tissue

JoVE 5209

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and changes in cell morphology. Studying these processes is essential to our understanding of nervous system function as well as our ability to diagnose and treat disorders that result from abnormal development. However, neural tissues are relatively inaccessible for experimental manipulations, especially in embryonic mammals. As a result, many scientists take advantage of explant culture in order to study neurodevelopmental processes in an “organotypic” environment, meaning that the tissue is removed from the organism but its complex cellular architecture is maintained. Generally, explant cultures are created by careful dissection of neural tissue that is then submerged in carefully designed growth media and cultured in vitro. This video will first provide a brief overview of neural explant culture, including its advantages over other in vitro methods and important considerations for maintaining healthy tissue. Next, a general protocol will be provided for setting up an explant culture from embryonic mouse brain, outlining the isolation of embryos from the mother and dissection of the brain. The presentation also includes an overview of slice culture


 Neuroscience

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Primary Neuronal Cultures

JoVE 5214

The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach is to generate a primary culture by dissecting nervous system tissue, dissociating it into single cells, and growing those cells in vitro. Primary cultures make neurons and glia easily accessible to the experimental tools required for techniques like genetic manipulation and time-lapse imaging. Furthermore, these cultures represent a highly controllable environment in which to study complex phenomena such as cell-cell interactions. This video provides an overview of the major steps in producing primary neuronal cultures, which include selecting and dissecting the tissue of interest, mechanically and chemically breaking down the tissue to produce a single cell suspension, plating the cells, and maintaining the cultures in the appropriate media. Several example experiments are also presented to show how cultured cells can be used to investigate protein trafficking, morphological changes, and electrophysiology in living neurons.


 Neuroscience

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An Introduction to Developmental Genetics

JoVE 5325

Development is the complex process through which a single-celled embryo transforms into a multicellular organism. Developmental processes are guided by information encoded in an organism\'s DNA, and geneticists are trying to understand how this information leads to a fully formed organism.

This video reviews seminal research in the field of developmental biology, including the identification of specific genes that control various embryonic processes. An introduction to the major questions asked by developmental geneticists, and the prominent methods used to answer them, is also provided. Finally, several applications of these prominent methods are discussed, in order to show specific experiments currently being performed in this field.


 Developmental Biology

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Gene Silencing with Morpholinos

JoVE 5326

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.

This video reviews proper experimental design when using these oligonucleotides. Following that, an explanation of morpholino microinjection techniques in zebrafish and the analysis of resulting phenotypes will be discussed. Finally, we showcase examples of specific applications where morpholino technology is used to model developmental disorders or to study tissue regeneration.


 Developmental Biology

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An Introduction to Molecular Developmental Biology

JoVE 5328

Molecular signals play a major role in the complex processes occurring during embryonic development. These signals regulate activities such as cell differentiation and migration, which contribute to the formation of specific cell types and structures. The use of molecular approaches allows researchers to investigate these physical and chemical mechanisms in detail.

This video will review a brief history of the study of molecular events during development. Next, key questions asked by molecular developmental biologists today will be reviewed, followed by a discussion of several prominent methods used to answer these questions, such as staining, explant culture, and live-cell imaging. Finally, we will look at some current applications of these techniques to the study of developmental biology.


 Developmental Biology

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Explant Culture for Developmental Studies

JoVE 5329

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo approach allows researchers to manipulate and observe developing tissues in ways that are not possible in vivo. Once established, explant culture is frequently used to understand the role of genes and signaling molecules in organogenesis. This video will first introduce the basic principles of explant culture and demonstrate a protocol to isolate and grow explanted mammalian tissues. Common genetic and molecular methods of manipulating explant cultures will then be discussed. Finally, the viewers will learn about how explant techniques are currently being applied to study organ development.


 Developmental Biology

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Whole-Mount In Situ Hybridization

JoVE 5330

Whole-mount in situ hybridization (WMISH) is a common technique used for visualizing the location of expressed RNAs in embryos. In this process, synthetically produced RNA probes are first complementarily bound, or "hybridized," to the transcripts of target genes. Immunohistochemistry or fluorescence is then used to detect these RNA hybrids, revealing spatial and temporal patterns of gene expression. Unlike traditional in situ hybridization techniques, which require thin tissue sections whose images will need to be computationally reassembled, the whole-mount technique allows gene expression patterns to be assessed over the entire embryo or structure. This video will introduce the basic concepts of whole mount staining and detail key procedural steps, including probe design and production, embryo fixation and staining, and post-hybridization signal detection. Viewers will then learn about how developmental biologists are applying WMISH to current research studies.


 Developmental Biology

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An Introduction to Stem Cell Biology

JoVE 5331

Cells that can differentiate into a variety of cell types, known as stem cells, are at the center of one of the most exciting fields of science today. Stem cell biologists are working to understand the basic mechanisms that regulate how these cells function. These researchers are also interested in harnessing the remarkable potential of stem cells to treat human diseases.

Here, JoVE presents an introduction to the captivating world of stem cell biology. We begin with a timeline of landmark studies, from the first experimental evidence for hematopoietic stem cells in the 1960s, to more recent breakthroughs like induced pluripotent stem cells. Next, key questions about stem cell biology are introduced, for example: How do these cells maintain their unique ability to undergo self-renewal? This is followed by a discussion of some prominent methods used to answer these questions. Finally, several experiments are presented to demonstrate the use of stem cells in regenerative medicine.


 Developmental Biology

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Embryonic Stem Cell Culture and Differentiation

JoVE 5332

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal and pluripotency. Stem cell biologists are continuously optimizing methods to improve the efficiency of ES cell culture, and are simultaneously trying to direct the differentiation of ES cells into specific cell types that could be used in regenerative medicine. This video describes the basic principles of ES cell culture, and demonstrates a general protocol to grow and passage ES cells. We also take a closer look at the hanging drop method, which is used to differentiate ES cells. Finally, this video will describe a few applications of ES cell culture and differentiation techniques, including a method used to generate functional heart muscle cells in vitro.


 Developmental Biology

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Induced Pluripotency

JoVE 5333

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem cells, iPSCs can be grown in culture conditions that promote differentiation into different cell types. Thus, iPSCs may provide a potentially unlimited source of any human cell type, which is a major breakthrough in the field of regenerative medicine. However, more research into the derivation and differentiation of iPSCs is still needed to actually use these cells in clinical practice. This video first introduces the fundamental principles behind cellular reprogramming, and then demonstrates a protocol for the generation of iPSCs from differentiated mouse embryonic fibroblasts. Finally, it will discuss several experiments in which scientists are improving or applying iPSC generation techniques.


 Developmental Biology

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An Introduction to Organogenesis

JoVE 5334

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying organogenesis want to better understand the genetic programs, cell-cell interactions, and mechanical forces involved in this process. Ultimately, scientists hope to use this knowledge to create therapies and artificial organs that will help treat human diseases. This video offers a comprehensive overview of organogenesis, starting with historical highlights describing the breakthrough studies done in the 1800\'s, all the way to the first human surgery using tissue-engineered organs performed in 2008. Next, key questions asked by developmental biologists are introduced, followed by a discussion of how tissue transplantations, imaging, and in vitro culture techniques can be used to answer these queries. Finally, we describe how these methods are currently being employed in developmental biology laboratories.


 Developmental Biology

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Fate Mapping

JoVE 5335

Fate mapping is a technique used to understand how embryonic cells divide, differentiate, and migrate during development. In classic fate mapping experiments, cells in different areas of an embryo are labeled with a chemical dye and then tracked to determine which tissues or structures they form. Technological improvements now allow for individual cells to be marked and traced throughout embryonic development and adulthood. This video reviews the concepts behind fate mapping, and then details a fate mapping protocol in zebrafish using photoactivatable fluorescent proteins. Finally, specific applications and modifications of this unique technique are discussed.


 Developmental Biology

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Transplantation Studies

JoVE 5336

Many developmental biologists are interested in the molecular signals and cellular interactions that induce a group of cells to develop into a particular tissue. To investigate this, scientists can use a classic technique known as transplantation, which involves tissue from a donor embryo being excised and grafted into a host embryo. By observing how transplanted tissues develop in host environments, scientists have started to dissect the molecular pathways underlying development. In this video, we first look at the role of cellular interactions in development, and move on to a basic transplantation protocol. Finally, some specific developmental studies utilizing this technique are discussed, which examine the effect of tissue transplantation on the fate of donor and host tissue.


 Developmental Biology

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Tissue Regeneration with Somatic Stem Cells

JoVE 5339

Somatic or adult stem cells, like embryonic stem cells, are capable of self-renewal but demonstrate a restricted differentiation potential. Nonetheless, these cells are crucial to homeostatic processes and play an important role in tissue repair. By studying and manipulating this cell population, scientist may be able to develop new regenerative therapies for injuries and diseases.

This video first defines somatic stem cells, and then explores the role these cells play in tissue regeneration. This is emphasized in a description of a protocol that isolates muscle satellite cells and uses them to repair muscle damage in a mouse model of muscular dystrophy. Finally, we discuss specific tissue regeneration studies utilizing somatic stem cells.


 Developmental Biology

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An Introduction to Motor Control

JoVE 5422

Motor control involves integration and processing of sensory information by our nervous system, followed by a response through our skeletal system to perform a voluntary or involuntary action. It is vital to understand how our neuroskeletal system controls motor behavior in order to evaluate injuries pertaining to general movement, reflexes, and coordination. An improved understanding of motor control will help behavioral neuroscientists in developing useful tools to treat motor disorders, such as Parkinson's or Huntington's disease. This video briefly reviews the neuroanatomical structures and connections that play a major role in controlling motion. Fundamental questions currently being asked in the field of motor control are introduced, followed by some of the methods being employed to answer those questions. Lastly, the application sections reviews a few specific experiments conducted in neuroscience labs interested in studying this phenomenon.


 Behavioral Science

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From Theory to Design: The Role of Creativity in Designing Experiments

JoVE 10047

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

Research studies come into being when a researcher speculates about human thought, emotions, or behavior, and has a theory that offers a potential explanation. Often the researcher’s theory is firmly situated in everyday common experiences that may not naturally lend themselves to direct empirical study. For example, researchers speculated that perception of a person on Facebook is influenced by the appearances and comments of the person’s Facebook friends.1 It is difficult to test this theory using real-life Facebook profiles. Instead, researchers must use their creativity to design a study—in this case, using fake profiles that look highly realistic—to test their theory.  This video demonstrates how researchers test a central tenet of a popular social psychology theory. Specifically, this video shows a test of whether engaging in a self-expanding activity leads a person to feel a greater sense of self-efficacy.2 Psychological studies often use higher sample sizes than studies in other sciences. A large number


 Experimental Psychology

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X-ray Fluorescence (XRF)

JoVE 5498

Source: Laboratory of Dr. Lydia Finney — Argonne National Laboratory

X-ray fluorescence is an induced, emitted radiation that can be used to generate spectroscopic information. X-ray fluorescence microscopy is a non-destructive imaging technique that uses the induced fluorescence emission of metals to identify and quantify their spatial distribution.


 Analytical Chemistry

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Using a pH Meter

JoVE 5500

Source: Laboratory of Dr. Zhongqi He - United States Department of Agriculture

Acids and bases are substances capable of donating protons (H+) and hydroxide ions (OH-), respectively. They are two extremes that describe chemicals. Mixing acids and bases can cancel out or neutralize their extreme effects. A substance that is neither acidic nor basic is neutral. The values of proton concentration ([H+]) for most solutions are inconveniently small and difficult to compare so that a more practical quantity, pH, has been introduced. pH was originally defined as the decimal logarithm of the reciprocal of the molar concentration of protons , but was updated to the decimal logarithm of the reciprocal of the hydrogen ion activity . The former definition is now occasionally expressed as p[H]. The difference between p[H] and pH is quite small. It has been stated that pH = p[H] + 0.04. It is common practice to use the term 'pH' for both types of measurements. The pH scale typically ranges from 0 to 14. For a 1 M solution of a strong acid, pH=0 and for a 1 M solution of a strong base, pH=14. Thus, measured pH values will lie mostly in the rang


 General Chemistry

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Cyclic Voltammetry (CV)

JoVE 5502

Source: Laboratory of Dr. Kayla Green — Texas Christian University

A Cyclic Voltammetry (CV) experiment involves the scan of a range of potential voltages while measuring current. In the CV experiment, the potential of an immersed, stationary electrode is scanned from a predetermined starting potential to a final value (called the switching potential) and then the reverse scan is obtained. This gives a 'cyclic' sweep of potentials and the current vs. potential curve derived from the data is called a cyclic voltammogram. The first sweep is called the 'forward scan' and the return wave is called the 'reverse scan'. The potential extremes are termed the 'scan window'. The magnitude of reduction and oxidation currents and the shape of the voltammograms are highly dependent on analyte concentration, scan rates, and experimental conditions. By varying these factors, cyclic voltammetry can yield information regarding the stability of transition metal oxidation state in the complexed form, reversibility of electron transfer reactions, and information regarding reactivity. This video will explain the basic setup for a cyclic voltammetry experiment including analyte preparation and setting up the electrochemical cell. A simple cyclic voltammetry experiment will be presented.


 Analytical Chemistry

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Detection of Bacteriophages in Environmental Samples

JoVE 10190

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Alex Wassimi

Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells. Viruses are ultramicroscopic—too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails. Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.


 Environmental Microbiology

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An Overview of Genetic Analysis

JoVE 5540

An organism’s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its parents. To varying degrees, genes interact with each other and environmental factors to generate traits. The distribution of alleles and traits within a population is influenced by a number of factors, including natural selection, migration, and random genetic drift.In this video, JoVE introduces some of the foundational discoveries in genetics, from Gregor Mendel’s elucidation of the genetic basis of inheritance, to how natural processes affect allele distributions within populations, to the modern synthesis of biology that brought together Mendelian genetics and Darwinian evolution. We then review the questions asked by geneticists today regarding how genes influence traits, and some of the main tools used to answer these questions. Finally, several applications of techniques such as genetic crosses, screens and evolution experiments will be presented.


 Genetics

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An Overview of Gene Expression

JoVE 5546

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer.

This video highlights important historical discoveries relating to gene expression, including the understanding of how distinct combinations of DNA bases encode the amino acids that make up proteins. Key questions in the field of gene expression research are explored, followed by a discussion of several techniques used to measure gene expression and investigate its regulation. Finally, we look at how scientists are currently using these techniques to study gene expression.


 Genetics

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Expression Profiling with Microarrays

JoVE 5547

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.


 Genetics

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Chromatin Immunoprecipitation

JoVE 5551

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as “scaffolds” around which DNA can be wrapped, forming a complex called “chromatin”. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to better understand which DNA regions associate with specific histone modifications or other gene regulatory proteins. Antibodies are used to isolate the protein of interest, and the bound DNA is extracted for analysis. Here, JoVE presents the principles behind ChIP, discussing specific histone modifications and their relationship to gene expression and DNA organization. We then review how to perform a ChIP protocol, and explore the ways scientists are currently using this technique.


 Genetics

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An Overview of Genetic Engineering

JoVE 5552

Genetic engineering – the process of purposefully altering an organism’s DNA – has been used to create powerful research tools and model organisms, and has also seen many agricultural applications. However, in order to engineer traits to tackle complex agricultural problems such as stress tolerance, or to realize the promise of gene therapy for treating human diseases, further advances in the field are still needed. Important considerations include the safe and efficient delivery of genetic constructs into cells or organisms, and the establishment of the desired modification in an organism’s genome with the least “off-target” effects. JoVE’s Overview of Genetic Engineering will present a history of the field, highlighting the discoveries that confirmed DNA as the genetic material and led to the development of tools to modify DNA. Key questions that must be answered in order to improve the process of genetic engineering will then be introduced, along with various tools used by genetic engineers. Finally, we will survey several applications demonstrating the types of experimental questions and strategies in the field today.


 Genetics

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Using Differential Scanning Calorimetry to Measure Changes in Enthalpy

JoVE 5559

Source: Laboratory of Dr. Terry Tritt — Clemson University

Differential Scanning Calorimetry (DSC) is a method of thermodynamic analysis based on heat-flux method, wherein a sample material (enclosed in a pan) and an empty reference pan are subjected to identical temperature conditions. The energy difference that is required to maintain both the pans at the same temperature, owing to the difference in the heat capacities of the sample and the reference pan, is recorded as a function of temperature. This energy released or absorbed is a measure of the enthalpy change (ΔΗ) of the sample with respect to the reference pan.


 General Chemistry

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Pelvic Exam II: Speculum Exam

JoVE 10141

Source:

Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT

Tiffany Cook, GTA, Praxis Clinical, New Haven, CT

Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT

Providing comfortable speculum placement is an important skill for providers to develop, since the speculum is a necessary tool in many gynecological procedures. Patients and providers are often anxious about the speculum exam, but it is entirely possible to place a speculum without patient discomfort. It's important for the clinician to be aware of the role language plays in creating a comfortable environment; for instance, a provider should refer to the speculum "bills" rather than "blades" to avoid upsetting the patient. There are two types of speculums: metal and plastic (Figure 1). This demonstration utilizes plastic, as plastic speculums are most commonly used in clinics for routine testing. When using a metal speculum, it's recommended to use a Graves speculum if the patient has given birth vaginally, and a Pederson speculum if the patient has not. Pederson and Graves speculums are different shapes, and both come in many different sizes (me


 Physical Examinations II

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An Introduction to Cell Division

JoVE 5640

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell organisms. In multicellular organisms, cell division contributes to growth, development, repair, and the generation of reproductive cells (sperms and eggs). Cell division is a tightly regulated process, and aberrant cell division can cause diseases, notably cancer. JoVE's Introduction to Cell Division will cover a brief history of the landmark discoveries in the field. We then discuss several key questions and methods, such as cell cycle analysis and live cell imaging. Finally, we showcase some current applications of these techniques in cell division research.


 Cell Biology

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An Introduction to Cell Motility and Migration

JoVE 5643

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues and organs during development, but on the other hand, the same mechanisms are used by tumor cells to move and spread in a process known as cancer metastasis. One of the primary cellular machineries that make cell movement possible is an intracellular network of myosin and actin molecules, together known as “actomyosin”, which creates a contractile force to pull a cell in different directions.In this video, JoVE presents a historical overview of the field of cell migration, noting how early work on muscle contraction led to the discovery of the actomyosin apparatus. We then explore some of the questions researchers are still asking about cell motility, and review techniques used to study different aspects of this phenomenon. Finally, we look at how researchers are currently studying cell migration, for example, to better understand metastasis.


 Cell Biology

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The Transwell Migration Assay

JoVE 5644

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants. This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.


 Cell Biology

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FM Dyes in Vesicle Recycling

JoVE 5648

FM dyes are a class of fluorescent molecules that has found important use in studying the vesicle recycling process. By virtue of a chemical structure, these molecules can insert themselves into the outer leaflet of phospholipid bilayer membranes. After membrane insertion, they are internalized into the cell via endocytosed vesicles, and released when these vesicles recycle back to the membrane. Since, these dyes fluoresce strongly in the hydrophobic environment within membranes and weakly in the extracellular compartment, FM fluorescence levels can be used to track vesicular activity throughout the recycling process.This video provides an introduction to the use of FM dyes in experiments aimed to examine vesicle recycling. We first review the biochemistry of FM dyes and how their properties permit their use in these experiments. We then go through a general protocol for using FM dyes in such studies, and finally, discuss some recent research that makes use of these unique molecules.


 Cell Biology

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Dialysis: Diffusion Based Separation

JoVE 5684

Dialysis is a common technique used in biochemistry for separating molecules based on diffusion. In this procedure, a semipermeable membrane allows the movement of certain molecules based on size. This method can be applied to the removal of buffer, known as desalting, or exchanging buffer molecules or ions from a protein solution.

This video covers the principles of dialysis along with a general procedure.  Several applications of dialysis are reviewed, including the removal of gradient reagents following ultracentrifugation, removing detergent after a membrane protein extraction, and the reconstitution of proteins by changing the solution environment. Biochemical samples typically have high buffer concentrations that can disrupt downstream processing and analysis. Dialysis is a common, inexpensive technique used to separate molecules based on diffusion. The method utilizes a semi-permeable membrane that allows the movement of certain components, based on size. This video will show the concepts of dialysis, a general procedure, and some of its uses in biochemistry. The most important aspect of dialysis is a semi-permeable membrane, which has pores that impose a molecular weight cut-off, allowing molecules below a certain size to pass through. For example, a 10k


 Biochemistry

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Density Gradient Ultracentrifugation

JoVE 5685

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. A high-speed ultracentrifuge is used to accelerate this process in order to separate biomolecules within a density gradient, which can be established by layering liquids of decreasing density in a centrifuge tube. The video will cover the principles of density gradient ultracentrifugation, including a procedure that demonstrates sample preparation, creation of a sucrose gradient, ultracentrifugation, and collection of fractionated analytes. The applications section discusses isolation of multi-protein complexes, isolation of nucleic acid complexes, and separation using cesium chloride density gradients. Density gradient ultracentrifugation is a common approach to isolate and purify cell structures for biochemical experiments. The technique uses a high-speed, or ultra, centrifuge to nondestructively separate cellular components in a density gradient. This video describes the principles of density gradient ultracentrifugation, provides a general procedure using a sucrose gradient, and discusses some applications.

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Biofuels: Producing Ethanol from Cellulosic Material

JoVE 10014

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol. The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast. Ethanol production is monitored using an ethanol probe. The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc. An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol. As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available. With an increased emphasis on ‘inquiry-based learning”, scientific probes are becoming more popular. Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions. Anothe


 Environmental Science

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Two-Dimensional Gel Electrophoresis

JoVE 5686

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights. The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM). Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a


 Biochemistry

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Metabolic Labeling

JoVE 5687

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR. This video introduces the concepts of metabolic labeling and show two general procedures.  The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos. Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications. Metabolic lab


 Biochemistry

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Tandem Mass Spectrometry

JoVE 5690

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a second mass spectrometer where the fragments are analyzed. This video introduces the principles of tandem mass spectrometry, including mass-to-ratio selection and dissociation methods. Also shown is a general procedure for analyzing a biochemical compound using tandem mass spectrometry with collision-induced dissociation. The applications section covers selection reaction monitoring, determination of protein post-translation modifications, and detection of tacrolimus levels in blood. Tandem mass spectrometry links together multiple stages of mass spectrometry to first isolate a biomolecule, and then determine aspects of its chemical makeup. Biomolecules have large, complex structures, making it difficult to determine their molecular composition. Tandem mass spectrometry selects a molecule of interest that is later fragmented into multiple subunits, which can help elucidate its identification and sequence. This video will show the


 Biochemistry

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