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Cold Spring Harbor Protocols

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https://www.readbyqxmd.com/read/30104411/lymphocyte-deficiency-induced-by-sublethal-irradiation-in-xenopus
#1
Louise A Rollins-Smith, Jacques Robert
In many studies of diseases affecting amphibians, it is important to determine to what extent lymphocyte-mediated defenses are involved. For example, in studies of the nature of the immune response of Xenopus laevis to the amphibian chytrid fungus, Batrachochytrium dendrobatidis , it was essential to determine if mucosal antimicrobial peptides or lymphocyte-mediated immunity was most important for resistance to this skin pathogen. In this protocol, we describe a method for sublethal irradiation to reduce lymphocyte numbers...
August 13, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30104410/mass-spectrometry-based-absolute-quantification-of-single-xenopus-embryo-proteomes
#2
Rik G H Lindeboom, Arne H Smits, Matteo Perino, Gert Jan C Veenstra, Michiel Vermeulen
Early Xenopus development is characterized by a poor correlation between global mRNA and protein abundances due to maternal mRNA and protein loading. Therefore, proteome profiling is necessary to study gene expression dynamics during early Xenopus development. In contrast to mammals, single Xenopus eggs and embryos contain enough protein to allow identification and quantification of thousands of proteins using mass spectrometry-based proteomics. In addition to investigating developmental processes, single egg or blastomere proteomes can be used to study cell-to-cell variability at an unprecedented depth...
August 13, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30042138/following-endocrine-disrupting-effects-on-gene-expression-in-xenopus-laevis
#3
Petra Spirhanzlova, Michelle Leemans, Barbara A Demeneix, Jean-Baptiste Fini
Endocrine-disrupting chemicals (EDCs), found in all categories of chemicals, are suspected to be a cause of declining well-being and human health, both as single molecules and as mixtures. It is therefore necessary to develop high throughput methods to assess the endocrine-disrupting potential of multiple chemicals currently on the market that are as yet untested. An advantage of in vivo chemical screening is that it provides a full spectrum of physiological impacts exerted by a given chemical. Xenopus laevis is an ideal model organism to test thyroid axis disruption in vivo as thyroid hormones (THs) are highly conserved across vertebrates and orchestrate tadpole metamorphosis...
July 24, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30042137/chip-sequencing-in-xenopus-embryos
#4
Saartje Hontelez, Ila van Kruijsbergen, Gert Jan C Veenstra
Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) is a powerful technique for mapping in vivo, genome-wide DNA-protein interactions. The interplay between DNA and proteins determines the transcriptional state of the genome. Using specific antibodies for the ChIP, it is possible to generate genome-wide profiles of histone posttranslational modifications, providing insight into the epigenetic memory and developmental potential of cells. The interactions between DNA and proteins involved in epigenetic regulation and transcription are highly dynamic during embryonic development...
July 24, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30042136/assay-for-transposase-accessible-chromatin-sequencing-using-xenopus-embryos
#5
Ann Rose Bright, Gert Jan C Veenstra
The DNA of eukaryotic genomes is packaged into chromatin by nucleosomes. This not only compacts the DNA but also plays a central role in gene regulation and establishment of cellular identity during development. Because of this packaging, the DNA is relatively inaccessible to nucleoplasmic factors; however, regulatory elements such as promoters, enhancers, and insulators are largely kept nucleosome-free. The assay for transposase-accessible chromatin (ATAC-seq) can be used to identify genomic locations of "open" chromatin, footprints of DNA-binding proteins, and positioned nucleosomes...
July 24, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30042135/assessing-antibody-responses-to-pathogens-or-model-antigens-in-xenopus-by-enzyme-linked-immunosorbent-assay-elisa
#6
Francisco De Jesús Andino, Jacques Robert
Xenopus laevis -specific monoclonal antibodies recognize IgM and IgY antibodies not only from X. laevis but also X. tropicalis as well as a variety of amphibian species including Ranidae , Bufonidae, and even some salamanders. These reagents are very useful to assess antibody responses from the serum or other animal secretions (e.g., peritoneal fluid). We present here an enzyme-linked immunosorbent assay (ELISA) optimized for amphibians that permits users to detect and titrate the presence of each type of antibody (IgM and IgY) produced against particular pathogens (e...
July 24, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29980588/adoptive-transfer-of-fluorescently-labeled-immune-cells-in-xenopus
#7
Kun Hyoe Rhoo, Jacques Robert
Adoptive cell transfer from inbred adult Xenopus to inbred tadpoles is a useful way to study the dissemination of immune cells or pathogen-infected immune cells in tadpoles. For example, Xenopus peritoneal leukocytes (PLs) can be readily infected by pathogens such as Frog virus 3 (FV3) and Mycobacterium marinum ( M. marinum ). By transferring fluorescently labeled, FV3-infected PLs into tadpoles, we observed infiltration of these cells into the tadpole's brain, which indicates that FV3-infected PLs can cross blood brain barrier...
July 6, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29980587/the-use-of-cell-free-xenopus-extracts-to-investigate-cytoplasmic-events
#8
Romain Gibeaux, Rebecca Heald
Experiments using cytoplasmic extracts prepared from Xenopus eggs have made important contributions to our understanding of the cell cycle, the cytoskeleton, and cytoplasmic membrane systems. Here we introduce the extract system and describe methods for visualizing and manipulating diverse cytoplasmic processes, and for assaying the functions, dynamics, and stability of individual factors. These in vitro approaches uniquely enable investigation of events at specific cell cycle states, including the assembly of actin- and microtubule-based structures, and the formation of the endoplasmic reticulum...
July 6, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895566/studies-of-limb-regeneration-in-larval-xenopus
#9
Anthony L Mescher, Anton W Neff
A basic protocol is given for animal maintenance and surgery in studies of hindlimb regeneration in larval Xenopus laevis Unlike urodele limbs, those of larval frogs typically show much more variation in the extent of regeneration after amputation. Such variation can be reduced by optimizing the conditions of larval maintenance to regulate the rates of growth and development, by selecting only larvae with normal rates of growth and morphological development for experimental use, and by attention to precision and consistency in the proximo-distal level of surgical amputation...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895565/chromatin-interaction-analysis-using-paired-end-tag-chia-pet-sequencing-in-tadpole-tissues
#10
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, Laurent M Sachs
Proper gene expression involves communication between the regulatory elements and promoters of genes. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity through DNA looping between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing is a powerful high-throughput method for detection of genome-wide chromatin interactions...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895564/investigating-the-cellular-and-molecular-mechanisms-of-wound-healing-in-xenopus-oocytes-and-embryos
#11
Jingjing Li, Enrique Amaya
The African clawed frog Xenopus has remarkable capacities to heal wounds rapidly and to regenerate complex tissues. Because of its experimental tractability, studies using Xenopus oocytes, embryos, and larvae have contributed extensively to our understanding of the molecular and cellular mechanisms underpinning wound healing and tissue regeneration. In this protocol, we describe wound-healing assays following mechanical or laser injuries of oocytes and multicellular epithelia in Xenopus laevis embryos. We also explain how to perform assays aimed at investigating the cellular and molecular events during wound healing, including gene knockdown and overexpression experiments...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895563/chromatin-immunoprecipitation-for-chromatin-interaction-analysis-using-paired-end-tag-chia-pet-sequencing-in-tadpole-tissues
#12
Nicolas Buisine, Xiaoan Ruan, Yijun Ruan, Laurent M Sachs
Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s)...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895562/methods-for-examining-lens-regeneration-in-xenopus
#13
Jonathan J Henry, Kimberly J Perry, Paul W Hamilton
Some vertebrates are able to regenerate the lens following its removal. This includes species in the genus Xenopus (i.e., X. laevis , X. tropicalis , and X. borealis ), the only anurans known to undergo lens regeneration. In Xenopus the regenerated lens is derived de novo from cells located within the basal-most layer of the larval corneal epithelium, and is triggered by factors provided by the neural retina. In larval frogs the corneal epithelium is underlain by an endothelium separated from the corneal epithelium except for a small central attachment (i...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29895561/ex-vivo-eye-tissue-culture-methods-for-xenopus
#14
Jonathan J Henry, Kimberly J Perry, Paul W Hamilton
Lens regeneration can be studied in whole animals following removal of the original lens (lentectomy). However, culturing a whole animal can be impractical for assays involving small molecule inhibitors or proteins. Ex vivo eye tissue culture is an alternative approach for examining lens regeneration. The ex vivo culture system offers certain advantages when compared to the in vivo regeneration assay, as the percentage of cases showing lens differentiation can exceed that seen in whole animals. This culture system also allows for the treatment of eye tissues in small volumes, which helps ensure reproducibility and reduces the amount (and cost) of small-molecule inhibitors or exogenous proteins, etc...
June 12, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29789402/rod-specific-ablation-using-the-nitroreductase-metronidazole-system-to-investigate-regeneration-in-xenopus
#15
Reyna I Martinez-De Luna, Michael E Zuber
Genetically controlled cell type-specific ablation provides a reproducible method to induce regeneration that can be temporally and spatially controlled. Until recently, regeneration studies in Xenopus have relied on surgical methods to stimulate regeneration. These methods are labor intensive and not as reproducible as a genetically controlled approach. In this protocol we describe selective ablation of rod photoreceptors in the premetamorphic Xenopus laevis retina using the nitroreductase/metronidazole (NTR/Mtz) system...
May 22, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30068592/generation-of-control-ligation-product-libraries-for-3c-analyses
#16
Tae Hoon Kim, Job Dekker
Control libraries are used to determine differences in the efficiency of polymerase chain reaction (PCR) primers in detecting 3C ligation products as well as any differences in the efficiency of amplification. Control libraries are generated by random ligation of restriction fragments so that each ligation product is present in equimolar amounts. This protocol begins with preparations of genomic DNA or BAC DNA preparations. The restriction enzyme used here should be the same as the one used for generation of the 3C or ChIP-loop library...
August 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30068591/generation-of-chip-loop-libraries
#17
Tae Hoon Kim, Job Dekker
The ChIP-loop method combines the standard 3C protocol with a routine ChIP protocol. This method allows the selective identification of long-range chromatin interactions between loci that are bound to specific proteins of interest. ChIP-loop experiments need to be designed and planned as described for the standard 3C protocol. First, identify a restriction enzyme that digests chromatin so that the elements of interest are located on separate restriction fragments. Second, determine whether an antibody is available that successfully immunoprecipitates the protein of interest in a typical ChIP experiment...
August 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30068590/generation-of-3c-libraries-from-cross-linked-cells
#18
Tae Hoon Kim, Job Dekker
This protocol, which describes how 3C libraries are made from formaldehyde-fixed mammalian cells, starts with cells that were previously cross-linked. Before embarking on a 3C-based analysis, decide which restriction enzyme will be used. Two parameters must be considered. First, the frequency with which a restriction enzyme cuts will determine the size of the restriction fragments and thus the resolution with which chromatin interactions can be detected. In most studies, researchers use restriction enzymes that cut once every ∼4 kb...
August 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30068589/altered-%C3%AE-lactamase-selection-approach-for-site-directed-mutagenesis
#19
Matteo Forloni, Alex Y Liu, Narendra Wajapeyee
Many protocols exist to perform site-directed mutagenesis, and here we present one of the more commonly used ones-site-directed mutagenesis by altered β-lactamase selection. β-Lactamase is an enzyme that cleaves ampicillin, rendering it impotent to bacteria. Certain mutations in the active site of β-lactamase can alter the substrate specificity of the enzyme and allow it to have increased hydrolytic activity for the cephalosporin family of antibiotics, a property not shared by wild-type lactamases. E. coli cells carrying the β-lactamase triple mutant G238S:E240:R241G show increased resistance to cefotaxime and ceftriaxone, two cephalosporins, compared with wild-type cells...
August 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/30068588/creating-insertions-or-deletions-using-overlap-extension-polymerase-chain-reaction-pcr-mutagenesis
#20
Matteo Forloni, Alex Y Liu, Narendra Wajapeyee
Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement...
August 1, 2018: Cold Spring Harbor Protocols
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