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Cold Spring Harbor Protocols

Susannah Rankin
Extracts prepared from the eggs of frogs, particularly Xenopus species, have provided critical material for seminal studies of nuclear and chromosome dynamics over several decades. Their usefulness for these types of analyses lies in several important characteristics: stockpiled nuclear components, absence of endogenous DNA, and intact and functioning signaling networks. These factors have allowed detailed molecular analyses of many aspects of chromosome biology, including DNA replication, checkpoint signaling, epigenetic control, and chromosome condensation, cohesion, and segregation...
August 27, 2018: Cold Spring Harbor Protocols
Lauren Wasson, Nirav M Amin, Frank L Conlon
Analysis of the molecular mechanisms driving cell specification, differentiation, and other cellular processes can be difficult due to the heterogeneity of tissues and organs. Therefore, it is critical to isolate pure cell populations in order to properly assess the function of certain cell types in the context of a tissue. This protocol describes use of the INTACT (isolation of nuclei tagged in specific cell types) method in Xenopus , followed by proteomics analysis of nuclear protein complexes. The INTACT protocol utilizes two transgenes: (1) a three-part nuclear targeting fusion (NTF) consisting of a nuclear envelope protein (Nup35) that targets the NTF to the nuclear membrane, an enhanced green fluorescent protein (EGFP) cassette for NTF visualization in live animals, and a biotin ligase receptor protein (BLRP) that provides a substrate for the biotinylation of the NTF, and (2) the E...
August 27, 2018: Cold Spring Harbor Protocols
Jin Sun Cho, Ira L Blitz, Ken W Y Cho
Transcriptional regulatory elements are typically found in relatively nucleosome-free genomic regions, often referred to as "open chromatin." Deoxyribonuclease I (DNase I) can digest nucleosome-depleted DNA (presumably bound by transcription factors), but DNA in nucleosomes or higher-order chromatin fibers is less accessible to the nuclease. The DNase-seq method uses high-throughput sequencing to permit the interrogation of DNase hypersensitive sites (DHSs) across the entire genome and does not require prior knowledge of histone modifications, transcription factor binding sites, or high quality antibodies to identify potentially active regions of chromatin...
August 21, 2018: Cold Spring Harbor Protocols
Yuuri Yasuoka, Masanori Taira
Introducing exogenous DNA into an embryo can promote misexpression of a gene of interest via transcription regulated by an attached enhancer-promoter. This protocol describes plasmid DNA microinjection into Xenopus embryos for misexpression of genes after zygotic gene expression begins. It also describes a method for coinjecting a reporter plasmid with mRNA or antisense morpholinos to perform luciferase reporter assays, which are useful for quantitative analysis of cis -regulatory sequences responding to endogenous or exogenous stimuli in embryos...
August 21, 2018: Cold Spring Harbor Protocols
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
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
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
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
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
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
Michael R Green, Joseph Sambrook
In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template...
October 1, 2018: Cold Spring Harbor Protocols
Edward A Greenfield, James DeCaprio, Mohan Brahmandam
Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. KLH-hapten conjugates are commonly used in antibody generation in a variety of hosts such as mice, rats, and rabbits...
October 1, 2018: Cold Spring Harbor Protocols
Larisa Litovchick
Electrophoretic transfer of proteins from gels to membranes can be achieved either by complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or by placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semidry transfer). For the wet transfer, the sandwich is placed in a buffer tank with platinum wire electrodes. For the semidry transfer, the gel-membrane sandwich is placed between carbon plate or stainless steel electrodes. Both methods are described here and generally work well, although semidry transfer is quicker and often more complete...
October 1, 2018: Cold Spring Harbor Protocols
Larisa Litovchick
This protocol describes Tris/glycine SDS-polyacrylamide gel electrophoresis, also known as SDS discontinuous gel electrophoresis or the Laemmli electrophoresis system. The gel-casting unit is assembled and tested to make sure that there are no leaks. Ammonium persulfate and tetramethylethylenediamine are added to the separating monomer solution, and the bottom (separating) gel is poured and allowed to polymerize. The top (stacking) gel is poured, and the comb is inserted to make the wells. The stacking gel is allowed to polymerize and the comb is then removed...
October 1, 2018: Cold Spring Harbor Protocols
Michael R Green, Joseph Sambrook
This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 µL (although most manufacturers recommend 50-µL reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. Discussion on preparing high-quality RNA, choosing a priming method, selecting an enzyme, and selecting an endogenous reference gene is also included...
October 1, 2018: Cold Spring Harbor Protocols
Michael R Green, Joseph Sambrook
There are few differences between the experimental steps necessary for amplifying template DNA in a real-time thermocycler and a standard polymerase chain reaction (PCR). In real-time PCR, it is necessary, however, to optimize the concentration of primers and probe and to perform a standard curve. It is also important to consider the data analysis method that will be used.
October 1, 2018: Cold Spring Harbor Protocols
Michael R Green, Joseph Sambrook
It is essential to prepare a standard curve for every real-time polymerase chain reaction (PCR) experiment. This protocol is used to construct a standard curve in which the template concentration is unknown. Such a standard curve is suitable for optimization experiments and for performing relative quantification by the standard curve method. To construct a standard curve for absolute quantification, the same principles apply as those presented here, but the concentration of the standards must be determined by an independent method...
October 1, 2018: Cold Spring Harbor Protocols
Michael R Green, Joseph Sambrook
Once primers and probes have been designed and obtained, it is necessary to optimize their concentration for each real-time polymerase chain reaction (PCR) assay. A set of PCRs is assembled in which the concentrations of forward and reverse primers are varied independently. Following amplification of the template DNA, amplification plots are compared. A standard curve is generated to determine the efficiency, sensitivity, and reproducibility of the assay. If SYBR Green I is used as the probe, then the melting curves are also analyzed...
October 1, 2018: Cold Spring Harbor Protocols
Allison Stewart
Mouse embryos and fetal organs have no pigmentation except for the retinal pigmented epithelium of the eye at subsequent stages of development. This makes it difficult to visualize and photodocument embryonic structures using conventional light microscopy. This protocol is used to localize specific proteins in whole embryos or fetal organs using immunofluorescence. Various imaging modalities can then be used to visualize these expression patterns, for example, confocal microscopy or optical projection tomography...
October 1, 2018: Cold Spring Harbor Protocols
Lisa Sandell, Kimberly Inman, Paul Trainor
Mouse embryos and fetal organs have no pigmentation except for the retinal pigmented epithelium of the eye at subsequent stages of development. This makes it difficult to visualize and photodocument embryonic structures using conventional light microscopy. A simple method is provided here that uses fluorescent nuclear stains. At the relatively low magnifications of dissecting microscopes, the nuclei of embryonic tissues essentially become "pixels" of fluorescence that "paint" the embryo, providing images similar to those obtained by low-magnification scanning electron microcopy (SEM)...
October 1, 2018: Cold Spring Harbor Protocols
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