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Methylated DNA Immunoprecipitation and Microarray-Based Analysis: Detection of DNA Methylation in Breast Cancer Cell LinesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA me...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Use of Reporter Genes to Study the Activity of Promoters in Ovarian Granulosa CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and β-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subu...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Use of Reporter Genes to Study Promoters of the Androgen ReceptorEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As a transcriptional regulator, the androgen receptor (AR) regulates the expression of many genes that are essential for male sexual differentiation, including the development of both normal prostate and prostate cancer. The AR acts by binding to regulatory DNA sequences found on the promoters of regulated genes. The study of AR activity on such responsive promoters is greatly facilitated by the use of the reporter gene assay, which provides a quantitative and reproducible method for studying the activity of such promoters. Among the several reporter genes that can be used, the genes encoding luciferase (Luc) and chloramph...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Isolation of Proteins Associated with the DNA-Bound Estrogen Receptor αEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Regulating gene expression is a complex process requiring the interaction of multiple transcription factors with their cognate recognition sequences. While these DNA-bound transcription factors are the primary drivers of gene expression, the capacity of a transcription factor to alter gene expression is tempered by its association with a host of coregulatory proteins that are recruited to the DNA-bound transcription factor. We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound estrogen receptor α (ERα) using an agarose-based electrophoretic mobility shift assay ...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Chromosome-Wide Analysis of Protein Binding and ModificationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications. This chapter present...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Genome-Wide Analysis for Protein−DNA Interaction: ChIP-ChipEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chromatin immunoprecipitation (ChIP) is a well-established procedure for protein−DNA interaction research. ChIP-chip, combining chromatin immunoprecipitation (ChIP) and microarray technology (Chip), enables scientists to survey genome-wide DNA binding sites for a given protein. The ChIP-chip technique has been used to identify transcription factor binding sites, explore epigenomic information and investigate factors in DNA replicate/repairs. Here we describe a protocol for ChIP-chip to study Pituitary Tumor Transforming Gene (PTTG1) in mammalian cells.
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Detection of ERα-SRC-1 Interactions Using Bioluminescent Resonance Energy TransferEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein−protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP2, a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET2 energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET2 assay as it is used to examine the physical interaction between the nuclear recep...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Detection of Proteins Sumoylated In Vivo and In VitroEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein−protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Consequently...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Identification of Alternative Transcripts Using Rapid Amplification of cDNA Ends (RACE)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for ...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Use of Laser Capture Microdissection in Studying Hormone-Dependent Diseases: EndometriosisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Endometriosis, a common gynecological disorder responsible for infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. Gene expression studies performed on endometriotic tissue homogenates have yielded results reflecting mRNA abundance in a mixture of cell types (including epithelial cells, stromal cells, fibrotic tissue, and muscle tissue). Therefore, a method for quantifying gene expression separately in individual cell populations is essential for identifying genetic markers. Laser capture microdissection is a technique for obtaining pure populations of cells ...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Determining the Affinity of Hormone−Receptor InteractionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Characterization of the binding of a hormone to its cognate receptor is a cornerstone of many studies in molecular and cellular endocrinology since this event represents the beginning of a specific cellular response, generally from a highly regulated extracellular messenger. The premise of hormone−receptor interaction follows from the law of mass action describing a reversible second-order reaction, hormone plus receptor, to give a non-covalently associated hormone-receptor complex. From this basic principle, a host of useful experimental parameters are available to the interested investigator. This chapter is focuse...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Reporter Mice for the Study of Intracellular Receptor ActivityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Real-Time Non-invasive Imaging of ES Cell-Derived Insulin Producing CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
ES cells are a potential source for insulin producing cells (IPCs). However, two major handicaps are establishing reliable differentiation protocols and the lack of imaging techniques that allow monitoring of these cells post-transplantation. Here, we describe a new approach for monitoring the in vitro differentiation and real-time, non-invasive imaging of ES cell-derived IPCs in vivo. ES cells were molecularly engineered so that the rat insulin promoter (RIP) driven luciferase (Luc) expression was specifically turned on and up regulated following their differentiation into IPCs. The rationale underlying this approach is t...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Transgenic Mouse Technology: Principles and MethodsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now referred to as transgenic mouse technology has revolutionized virtually all fields of biology and provided new genetic approaches to model many human diseases in a whole animal context. Several hundreds of transgenic lines with expression of foreign genes specifically targeted to desired organelles/cells/tissues have been characterized. Further, the ability to spatio-temporally inactivate or activate gene expression in vivo using the “Cre-lox” ...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Breast Tumor-Initiating Cells Isolated from Patient Core Biopsies for Study of Hormone ActionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In recent years, evidence has emerged supporting the hypothesis that cancer is a stem cell disease. The cancer stem cell field was led by the discovery of leukemia stem cells (Tan, B.T., Park, C.Y., Ailles, L.E., and Weissman, I.L. (2006) The cancer stem cell hypothesis: a work in progress. Laboratory Investigation. 86, 1203–1207), and within the past few years cancer stem cells have been isolated from a number of solid tumor including those of breast and brain cancer among others (Al-Hajj M., Wicha M.S., Benito-Hernandez A., Morrison, S.J., and Clarke, M.F. (2003) Prospective identification of tumorigenic breast can...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Markers of Oxidative Stress and Sperm Chromatin IntegrityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Oxidative stress (OS) is an imbalance between the amount of reactive oxygen species (ROS) produced and the ability of the antioxidants to scavenge these. OS has been established as a major etiological cause of male infertility. High levels of ROS are harmful and cause damage to sperm nuclear DNA. Evaluation of OS-related damage to spermatozoa is therefore highly relevant in assisted reproductive techniques (ART) such as intracytoplasmic sperm injection (ICSI). ICSI is an effective therapy for severe male factor infertility that bypasses the majority of reproductive tract deficiencies. Despite the controversial findings in ...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Planning and Executing a Genome Wide Association Study (GWAS)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In recent years, genome-wide association approaches have proven a powerful and successful strategy to identify genetic contributors to complex traits, including a number of endocrine disorders. Their success has meant that genome wide association studies (GWAS) are fast becoming the default study design for discovery of new genetic variants that influence a clinical trait or phenotype. This chapter focuses on a number of key elements that require consideration for the successful conduct of a GWAS. Although many of the considerations are common to any genetic study, the greater cost, extreme multiple testing, and greater op...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Determination of Serum Estradiol Levels by Radiometric and Chemiluminescent TechniquesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The ability to precisely measure circulating levels of hormones is a foundation of modern endocrinology. For assisted reproductive technologies such as in vitro fertilization (IVF), accurate determination of circulating levels of estradiol are crucial for patient management, retrieval of fertilizable oocytes, and successful pregnancy outcome. For many years, circulating levels of estradiol were determined by radioimmunoassay. More recently, nonradioactive techniques such as ELISAs or chemiluminescent approaches have replaced traditional radioimmunoassays. In the current chapter, we outline the procedures for analysis of ci...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Identification of Natural Human Glucocorticoid Receptor (hGR) Mutations or Polymorphisms and Their Functional Consequences at the Hormone–Receptor Interaction LevelEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Glucocorticoids regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress-related homeostasis. At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor α (hGRα), a ligand-dependent transcription factor ubiquitously expressed in almost all tissues and cells. The molecular mechanisms of hGRα action involve (a) binding to glucocorticoids, (b) cytoplasmic to nuclear translocation, (c) binding/association to DNA/chromatin, and (d) transcriptional activation or repression by interacting w...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Monitoring Insulin-Stimulated Production of Signaling Lipids at the Plasma MembraneEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lipid second messengers play important roles in many cell signaling cascades. Lipid signaling molecules allow for high specificity, rapid transduction, and rapid reversibility of localized stimulation events. Fluorescent sensors capable of detecting individual signaling lipids enable their production and degradation to be followed, revealing the nature and dynamics of signaling pathways. The following sections outline a method for using lipid sensors to monitor the production of signaling lipids on the plasma membrane of C2C12 myotubes in response to insulin signaling.
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Gene Expression Profiling in the Aging OvaryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA microarray is an important discovery technology that allows the analysis of the expression of thousands of genes at a time. Data from DNA microarrays elucidate fundamental biological processes through discovery of differential expression of genes not previously known or predicted to be involved in a particular process. In the ovary and other hormone-responsive tissues, the technology can be used to examine the effects of gene mutations, pharmaceutical agents, disease, hormones, developmental changes, or changes in gene expression related to aging.
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Genomics Analysis: EndometriumEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microarray technology has been used widely in gynecology. Numerous studies have used this method to address biological questions related to human endometrium. The cyclic changes of endometrium confer special characteristics that should be considered before genomic analysis. The present study reviews these considerations and the principles of transcriptomic analysis through an example of a comparison of three different phases of the menstrual cycle.
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Detection of Ovarian Matrix Metalloproteinase mRNAs by In Situ HybridizationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In situ hybridization represents a powerful technique to localize DNA or RNA of interest at the chromosomal or cellular level. In endocrine tissues composed of diverse and varied cell types, in situ hybridization has allowed the identification of specific cells responsible for the expression of genes controlling the function of the tissue. Our laboratory has routinely used this approach to understand the cellular expression of genes associated with the growth of the ovarian follicle, rupture of the follicle, and transformation of the ruptured follicle into the corpus luteum. The current study outlines the procedural detail...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Adenoviral Gene Transfer into Isolated Pancreatic IsletsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here in detail a protocol for adenoviral gene transfer into isolated mouse islets of the pancreas. Effective gene transfer into pancreatic islets using recombinant adenoviruses can be achieved with a multiplicity of infection (MOI) of 10. However, if the islets are not dispersed, adenoviral gene transfer is limited only to the cells on the periphery of the islets, which represent the glucagon-producing alpha cells in rodents. Dispersion of pancreatic islets with EGTA increases the efficiency of gene transfer into the cells within the core of the islets, which consist of insulin-producing beta cells.
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Basic Molecular Techniques for the Detection of Single Nucleotide Polymorphisms: Genome-Wide Applications in Search for Endocrine Tumor Related GenesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The necessity of genotyping high number of variations in extended sample sets has become apparent in the era of large genomic studies of common complex disorders, in cancer and in pharmacogenomics. The single nucleotide polymorphisms’ (SNPs) apparent advantages over other genetic markers such as high frequency, relative stability, and statistically random distribution across the genome have made them a method of choice for most of these genome-wide oriented applications. The requirement for simultaneous genotyping of high number of SNPs, keeping at the same time reasonable price and reliable accuracy, triggered the r...
Source: Springer protocols feed by Molecular Medicine - August 1, 2009 Category: Molecular Biology Source Type: info

Electroporation-Mediated Gene Transfer to the Developing Mouse Inner EarEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The mammalian inner ear forms from a thickened patch of head ectoderm called the otic placode. The placodal ectoderm invaginates to form a cup whose edges cinch together to establish a fluid-filled sac called the otic vesicle or otocyst. The progenitor cells lining the otocyst lumen will give rise to sensory and non-sensory cells of the inner ear. These formative stages of inner ear development are initiated during the first week of postimplantation embryonic development in the mouse. The inaccessibility of the inner ear in utero has hampered efforts to gain insight into the molecular mechanisms regulating essential develo...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Isolation of Sphere-Forming Stem Cells from the Mouse Inner EarEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

The Practical Use of Cre and loxP Technologies in Mouse Auditory ResearchEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene manipulation, specifically in the hair cells of the inner ear during development and adulthood in mice, is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. The Cre–loxP system has been widely used for such purposes. Many laboratories, including ours, have developed and characterized transgenic mouse lines that express or induce Cre activity specifically in inner ear hair cells. These Cre lines have been used with high...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Helios® Gene Gun–Mediated Transfection of the Inner Ear Sensory EpitheliumEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Helios® Gene Gun–mediated transfection is a biolistic method for mechanical delivery of exogenous DNA into cells in vitro or in vivo. The technique is based on bombardment of a targeted cellular surface by micron- or submicron-sized DNA-coated gold particles that are accelerated by a pressure pulse of compressed helium gas. The main advantage of Helios® Gene Gun–mediated transfections is that it functions well on various cell types, including terminally differentiated cells that are difficult to transfect, such as neurons or inner ear sensory hair cells, and cells in internal cellular layers, such as ne...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Lineage Analysis of Inner Ear Cells Using Genomic Tags for Clonal IdentificationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To understand the mechanisms of development of the inner ear, it is important to know the lineal relationships among the different cell types and the migrational boundaries of individual clones within the inner ear. This chapter details the basic methods for performing lineage analysis of the inner ear using replication-defective retroviral vectors in chicken embryos. Protocols are provided for generating avian retroviruses pseudotyped with vesicular stomatitis virus (VSV) envelopes to improve infectivity in early embryos. Moreover, we include the pioneering methods of the Cepko laboratory, whereby a library of DNA tags wa...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Genetic Fate-Mapping Approaches: New Means to Explore the Embryonic Origins of the Cochlear NucleusEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Greatly impacting the field of neural development are new technologies for generating fate maps in mice and thus for illuminating relationships between embryonic and adult brain structures. Until now, efforts in mammalian models such as the mouse have presented challenges because their in utero development limits the access needed for traditional methods involving tracer injection or cell transplantation. But access is no longer an obstacle. It is now possible to deliver cell lineage tracers via noninvasive genetic, rather than physical, means. The hinge-pin of these new “genetic fate mapping” technologies is a...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Atomic Force Microscopy in Studies of the CochleaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

In situ Hybridization Approach to Study mRNA Expression and Distribution in Cochlear Frozen SectionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In situ hybridization is well suited to obtaining specific topological information on gene transcripts and thereby to relating such observations to a particular function. In spite of the technical and practical difficulties, the application of molecular biological techniques such as in situ hybridization to the cochlea can provide important insights. However, the rarity of gene products (mRNA and proteins) in the cochlea and its fragile structure require the refinement and adaptation of in situ hybridization methods. The present chapter provides a detailed in situ hybridization protocol adapted to frozen tissue sections co...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Fluorescence Microscopy Methods in the Study of Protein Structure and FunctionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Ion Imaging in the Cochlear Hair CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Regulation of important cellular functions via signaling pathways is a fundamental property of the cell. Intracellular Ca2+ is probably a best known second messenger in cell biology. In mechanosensory cells of the inner ear, the hair cells, intracellular Ca2+ participates in a variety of functions including mechano-electrical transduction, synaptic transmission, and efferent regulation of the outer hair cells, one of two types of hair cells in the mammalian cochlea. The outer hair cells are responsible for the amplification of sound-induced vibrations within the cochlea, which determines the sensitivity of mammalian hearin...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Multiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory ResearchEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a compl...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Proteins in Inner-Ear Sensory EpitheliaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have uti...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Synthesis of Biotin-Labeled RNA for Gene Expression Measurements Using Oligonucleotide ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA po...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

In Vivo Verification of Protein Interactions in the Inner Ear by CoimmunoprecipitationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Genomics has provided us with vast amounts of data and thus, the challenge to identify and characterize gene products. Proteomics analysis, using methods such as yeast two-hybrid screenings, isoelectric focusing, and mass spectroscopy, generate potentially useful information. To determine functional relationships between and among proteins, however, the initial data for putative protein interactions must first be validated. One technique, which is considered the gold standard, is coimmunoprecipitation.
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Identification of Transcription Factor–DNA Interactions Using Chromatin Immunoprecipitation AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study tra...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

The Use of 2-D Gels to Identify Novel Protein–Protein Interactions in the CochleaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Functional proteomics comprises a wide range of technologies for the identification of novel protein-protein interactions and biological markers. Studies of protein-protein interactions have gained from the development of techniques and technologies such as immunoprecipitation, preparative two-dimensional (2-D) gel electrophoresis for peptide mass fingerprinting (PMF), using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These applications enabled the discovery of putative protein partners without a priori knowledge of...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Identification of Functionally Important Residues/Domains in Membrane Proteins Using an Evolutionary Approach Coupled with Systematic Mutational AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Structure-function studies of membrane proteins present a unique challenge to researchers due to the numerous technical difficulties associated with their expression, purification and structural characterization. In the absence of structural information, rational identification of putative functionally important residues/regions is difficult. Phylogenetic relationships could provide valuable information about the functional significance of a particular residue or region of a membrane protein. Evolutionary Trace (ET) analysis is a method developed to utilize this phylogenetic information to predict functional sites in prote...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Twist-Off Purification of Hair BundlesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here the “twist-off” method of bundle isolation, where dissected inner-ear organs are embedded in agarose, then subjected to a mechanical disruption that shears off bundles and leaves them in agarose blocks. With care in the dissection and in clean-up of the isolated bundles, contamination from cell bodies can be kept to a minimum. Isolated bundles can be analyzed by a variety of techniques, including immunocytochemistry, SDS-PAGE, immunoblotting, and mass spectrometry.
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Yeast Two-Hybrid Screening to Test for Protein–Protein Interactions in the Auditory SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a protocol to screen for protein-protein interactions using the Gal-4 based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method to gives the most number of true interactions. Where a pre-made library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick and...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Positional Cloning of Deafness GenesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The identification of the majority of the known causative genes involved in nonsyndromic sensorineural hearing loss (NSHL) started with linkage analysis as part of a positional cloning procedure. The human and mouse genome projects in combination with technical developments on genotyping, transcriptomics, proteomics, and the creation of animal models have greatly enhanced the speed of disease gene identification. In the present chapter, we first discuss the possibilities for exclusion of known NSHL loci and genes. Subsequently, methods are described to determine the genomic regions that contain the genetic defects. These i...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Fluorescence “In Situ” Hybridization for the Detection of Biofilm in the Middle Ear and Upper Respiratory Tract MucosaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Most chronic bacterial infections are associated with biofilm formation wherein the bacteria attach to mucosal surfaces, wound tissue, or medical device surfaces in the human body via the formation of an extracellular matrix. Biofilms assume complex three-dimensional structures dependent on the species, the strain, and the prevailing environmental conditions and are composed of both the bacteria and the extracellular slime-like matrices, which surround the bacteria. Bacteria deep in the biofilm live under anaerobic conditions and must use alternatives to O2 as a terminal electron acceptor. Thus, the metabolic rates of thes...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Multilocus Sequence Typing and Pulsed Field Gel Electrophoresis of Otitis Media Causing PathogensEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the three leading bacteria species associated with otitis media. Defining the molecular epidemiology of bacteria known to cause otitis media is of great importance, in both clinical and research settings. PFGE and MLST provide data for the characterization of isolates’ genetic relatedness, yet they differ in the types of studies for which they are most useful. Consequently, knowledge of both techniques is important for laboratories intending to study the molecular epidemiology of otitis media–associated bacterial pathogens.
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

Molecular Biology of Vestibular SchwannomasEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recent advances in molecular biology have led to a better understanding of the etiology of vestibular schwannomas. The underlying purpose of vestibular schwannoma research is the development of new treatment options; however, such options have not yet been established. A fundamental understanding of the underlying molecular events leading to tumor formation began when mutations in the neurofibromatosis type 2 (NF2) tumor suppressor gene were identified in vestibular schwannomas. The clinical characteristics of vestibular schwannomas and neurofibromatosis type 2 (NF2) syndromes have both been related to alterations in the N...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

RNA Isolation from Xenopus Inner Ear Sensory Endorgans for Transcriptional Profiling and Molecular CloningEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microar...
Source: Springer protocols feed by Molecular Medicine - October 24, 2008 Category: Molecular Biology Source Type: info

In Vitro Transdifferentiation of Human Hepatoma Cells into Pancreatic-Like CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transdifferentiation is defined as an irreversible switch in postnatal life of one differentiated cell to another. Transdifferentiation from different cellular origins into pancreatic-like β-cells is of clinical significance since this approach may offer a potential cure for diabetes. In order to achieve this goal, the liver is considered as a suitable candidate due to its close developmental relationship to the pancreas, its large size and a well-documented regenerative capacity that could provide enough original tissues to initiate the transdifferentiation procedure. In this chapter, we describe a protocol to overex...
Source: Springer protocols feed by Molecular Medicine - August 1, 2008 Category: Molecular Biology Source Type: info