ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.
21 Related JoVE Articles!
Development of a Virtual Reality Assessment of Everyday Living Skills
Institutions: NeuroCog Trials, Inc., Duke-NUS Graduate Medical Center, Duke University Medical Center, Fox Evaluation and Consulting, PLLC, University of Miami Miller School of Medicine.
Cognitive impairments affect the majority of patients with schizophrenia and these impairments predict poor long term psychosocial outcomes. Treatment studies aimed at cognitive impairment in patients with schizophrenia not only require demonstration of improvements on cognitive tests, but also evidence that any cognitive changes lead to clinically meaningful improvements. Measures of “functional capacity” index the extent to which individuals have the potential to perform skills required for real world functioning. Current data do not support the recommendation of any single instrument for measurement of functional capacity. The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is a novel, interactive gaming based measure of functional capacity that uses a realistic simulated environment to recreate routine activities of daily living. Studies are currently underway to evaluate and establish the VRFCAT’s sensitivity, reliability, validity, and practicality. This new measure of functional capacity is practical, relevant, easy to use, and has several features that improve validity and sensitivity of measurement of function in clinical trials of patients with CNS disorders.
Behavior, Issue 86, Virtual Reality, Cognitive Assessment, Functional Capacity, Computer Based Assessment, Schizophrenia, Neuropsychology, Aging, Dementia
P50 Sensory Gating in Infants
Institutions: University of Colorado School of Medicine, Colorado State University.
Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating.
Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e.
the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits.
Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.
Behavior, Issue 82, Child Development, Psychophysiology, Attention Deficit and Disruptive Behavior Disorders, Evoked Potentials, Auditory, auditory evoked potential, sensory gating, infant, attention, electrophysiology, infants, sensory gating, endophenotype, attention, P50
Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network
Institutions: Beth Israel Deaconess Medical Center.
The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3
It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6
and autism spectrum disorders 7
whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8
). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.
Neuroscience, Issue 46, Transcranial Magnetic Stimulation, rTMS, fMRI, Default Mode Network, functional connectivity, resting state
Assessment of Social Interaction Behaviors
Institutions: Mount Sinai Hospital, Mount Sinai Hospital, University of Toronto, University of Toronto, University of Toronto.
Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster
, Caenorhabditis elegans
, Mus musculus
, Rattus norvegicus
) have identified genes involved in the regulation of social behavior 2
. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3
). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7
). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7
. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7
. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.
Neuroscience, Issue 48, Mice, behavioral test, phenotyping, social interaction
A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
Institutions: Rosalind Franklin University of Medicine and Science, Rosalind Franklin University of Medicine and Science.
Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo
system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.
Medicine, Issue 72, Genetics, Genomics, Physiology, Anatomy, Biomedical Engineering, Metabolomics, Zebrafish, diabetes, metabolic memory, tissue regeneration, streptozocin, epigenetics, Danio rerio, animal model, diabetes mellitus, diabetes, drug discovery, hyperglycemia
A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H
]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Isolation of Human Islets from Partially Pancreatectomized Patients
Institutions: University Hospital Carl Gustav Carus, University of Technology Dresden, Paul Langerhans Institute Dresden, University Hospital Carl Gustav Carus, University of Technology Dresden.
Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1
have been hampered by the limited availability of type 2 diabetic islets from organ donors2
. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (>15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3
at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2
at 37 °C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.
Medicine, Issue 53, human islets, Diabetes mellitus, partial pancreatectomy, human islet isolation
A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice
Institutions: University of California, San Diego , San Diego VA Healthcare System.
The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3
H or 14
C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 μl of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e.
doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.
Medicine, Issue 75, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Nephrology, Kidney Function Tests, Glomerular filtration rate, rats, mice, conscious, creatinine, inulin, Jaffe, hypertension, HPLC, animal model
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis
Institutions: University of Colorado Boulder, NeuroScript LLC, University of California, San Diego.
Growing evidence suggests that movement abnormalities are a core feature of psychosis. One marker of movement abnormality, dyskinesia, is a result of impaired neuromodulation of dopamine in fronto-striatal pathways. The traditional methods for identifying movement abnormalities include observer-based reports and force stability gauges. The drawbacks of these methods are long training times for raters, experimenter bias, large site differences in instrumental apparatus, and suboptimal reliability. Taking these drawbacks into account has guided the development of better standardized and more efficient procedures to examine movement abnormalities through handwriting analysis software and tablet. Individuals at risk for psychosis showed significantly more dysfluent pen movements (a proximal measure for dyskinesia) in a handwriting task. Handwriting kinematics offers a great advance over previous methods of assessing dyskinesia, which could clearly be beneficial for understanding the etiology of psychosis.
Behavior, Issue 81, Schizophrenia, Disorders with Psychotic Features, Psychology, Clinical, Psychopathology, behavioral sciences, Movement abnormalities, Ultra High Risk, psychosis, handwriting, computer tablet, dyskinesia
gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair
Institutions: Centre Georges-François Leclerc.
The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM
, and TP53
) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.
Genetics, Issue 92, gDNA enrichment, Nextera, NGS, DNA damage, BRCA1, BRCA2
Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice
Institutions: Fujita Health University, Core Research for Evolutionary Science and Technology (CREST), National Institutes of Natural Sciences.
The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at https://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.
Behavior, Issue 85, Fear, Learning, Memory, ImageFZ program, Mouse, contextual fear, cued fear
Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
Institutions: Medical School Düsseldorf, Germany, University of Düsseldorf.
Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (Aβ, tau), Parkinson's Disease (α-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e.
the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2
, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3
. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro
revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4
, and that DISC1 aggresomes generated in vitro
, similar to what had been shown for Aβ6
, or SOD1 aggregates12
. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo
when stereotactically injected into the brain of recipient animals.
Molecular Biology, Issue 66, Neuroscience, Medicine, Genetics, Protein aggregate, aggresome, cell invasiveness, protein conformational disease, DISC1, DISC1opathy, purification, recombinant protein, multimerization, protein labeling, brain, rat, neuroscience
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Training Synesthetic Letter-color Associations by Reading in Color
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
A Rapid Technique for the Visualization of Live Immobilized Yeast Cells
Institutions: Princeton University.
We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also Xu et al., Mol. Cell 2006.
Microbiology, Issue 1, yeast, HML, HMR, epigenetic, loci, silencing, cerevisiae
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing