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丁香实验推荐阅读
Generating Transgenic Frog Embryos by Restriction Enzyme Mediated Integration (REMI)

Here we present a protocol for generating transgenic embryos in Xenopus laevis and Xenopus tropicalis. The method includes three steps: (1) The preparation of high-speed egg extracts, which facilitates the replacement of protamines in sperm nuclei with nucleosomes and decondenses t ...

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A Simple Method of Transgenesis Using I-Sce I Meganuclease in Xenopus

Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for gene ...

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Using C31 Integrase to Mediate Insertion of DNA in Xenopus Embryos

The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These metho ...

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Comparative Genomics-Based Identification and Analysis of Cis-Regulatory Elements

Identification of cis-regulatory elements, such as enhancers and promoters, is very important not only for analysis of gene regulatory networks but also as a tool for targeted gene expression experiments. In this chapter, we introduce an easy but reliable approach to predict enhancers of a g ...

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Xenopus Transgenics: Methods Using Transposons

The generation of transgenic animals is an essential tool for many genetic strategies. DNA “cut-and-paste” transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenop ...

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Tet-On Binary Systems for Tissue-Specific and Inducible Transgene Expression

Tissue-specific and inducible control of transgene expression is a cornerstone of modern studies in developmental biology. Even though such control of transgene expression has been accomplished in Xenopus, no general or widely available set of transgenic lines have been produced a ...

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Chromatin Immunoprecipitation Analysis of Xenopus Embryos

Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the ep ...

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Transcriptomics Using Next Generation Sequencing Technologies

Next generation sequencing technologies may now be applied to the study of transcriptomics. RNA-Seq or RNA sequencing employs high-throughput sequencing of complementary DNA fragments delivering a transcriptional profile. In this chapter, we aim to provide a starting point for Xe ...

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Databases of Gene Expression in Xenopus Development

Gene expression data for Xenopus are collected and curated in diverse forms and locations. The intention of this chapter is to give the reader a guide to the publicly accessible databases where these data can be found and an idea of the current scope and limitations of the data in these resources. Instru ...

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Investigating Alternative RNA Splicing in Xenopus

Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expr ...

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Immunoisolation of Protein Complexes from Xenopus

The immunoaffinity isolation of protein complexes is an essential technique for the purification and �concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laev ...

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Complementary Proteomic Analysis of Protein Complexes

Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we d ...

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Multicolor Fluorescent In Situ mRNA Hybridization (FISH) on Whole Mounts and Sections

In situ hybridization involves the hybridization of an antisense RNA probe to an mRNA transcript and it is a powerful method for the characterization of gene expression in tissues, organs, or whole organisms. Performed as a whole mount (WISH), it allows the detection of mRNA transcripts in three d ...

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Antibody Development and Use in Chromogenic and Fluorescent Immunostaining

Antibody-based detection of protein distribution patterns both in wholemount and on sections revolutionized Xenopus research and ushered in the visual-based era of Xenopus data presentation. The ability to view the distribution of a gene product throughout an embryo makes it possi ...

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FISH on 3D Preserved Bovine and Murine Preimplantation Embryos

Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of whole chromosomes or subchromosomal regions, such as chromosome arms, bands, centromeres, or single gene loci. FISH is routinely performed on chromosome spreads, as well as on three-dimens ...

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Fluorescence In Situ Hybridization on Early Porcine Embryos

Insight into the normal and abnormal function of an interphase nucleus can be revealed by using fluorescence in situ hybridization (FISH) to determine chromosome copy number and/or the nuclear position of loci or chromosome territories. FISH has been used extensively in studies of mouse a ...

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Fluorescence in situ Hybridization (FISH) for Genomic Investigations in Rat

This chapter concentrates on the use of fluorescence in situ hybridization (FISH) for genomic investigations in the laboratory rat (Rattus norvegicus). The selection of protocols included in the chapter has been inspired by a comprehensive range of previously published molecular c ...

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Three-Dimensional Fluorescence In Situ Hybridization in Mouse Embryos Using Repetitive Probe Sequences

A common problem in research laboratories that study the mammalian embryo is the limited supply of live material. For this reason, new methods are constantly being developed and existing methods for in vitro models using cells in culture are being adapted to represent embryogenesis. Three- ...

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Fluorescence in situ Hybridization with Bacterial Artificial Chromosomes (BACs) to Mitotic Heterochromatin of Drosophila

The organization of eukaryotic chromosomes into euchromatin and heterochromatin represents an enigmatic aspect of genome evolution. Constitutive heterochromatin is a basic, yet still poorly understood component of eukaryotic genomes and its molecular characterizati ...

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FISH on Chromosomes Derived from the Snail Model Organism Biomphalaria glabrata

The application of fluorescence in situ hybridization (FISH) for the mapping of single copy genes onto homologous chromosome has been integral to vast number genome sequencing projects, such as that of mouse and human. The chromosomes of these organisms are well-studied and are the staple re ...

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