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Genome-Wide Co-Localization Screening of Nuclear Body Components Using a Fluorescently Tagged FLJ cDNA Clone Library
Mammalian cell nuclei contain multiple granular structures, which are termed nuclear bodies. These structures are involved in various molecular events in the nucleus; they provide platforms for biogenesis of macromolecular complexes that are essential for gene expression, such as the ribosome and spliceosome; they act as reservoirs of various regulatory factors; and they are involved in the regulation of specific gene loci. Nuclear bodies are usually visualized by immunostaining for specific marker proteins. Although each type of nuclear body contains a distinct set of proteins, the protein components of most types of nucl...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Analyses of Nuclear Proteins and Nucleic Acid Structures Using Atomic Force Microscopy
Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid–protein complexes, reconstituted chromatin, the cel...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Ultrastructural Analysis of Nuclear Bodies Using Electron Microscopy
Recent immunofluorescent (IF) studies have discovered a variety of nuclear foci that have no known ultrastructurally defined counterpart. Using antibodies as ligands, immuno-electron microscopy (I-EM) is the method of choice for high-resolution recognition of these newly described nuclear compartments. However, noncoding RNAs (ncRNAs) have also been shown to be frequent components, sometimes essential, of nuclear bodies so that electron microscopic in situ hybridization (EM-ISH) can be used as an alternative means to characterize nuclear foci at the EM level. Among the array of protocols available, Lowicryl embedding of ch...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Live CLEM Imaging to Analyze Nuclear Structures at High Resolution
Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called “Live CLEM.” In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed us...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Visualization of Nucleic Acids with Synthetic Exciton-Controlled Fluorescent Oligonucleotide Probes
Engineered probes to adapt new photochemical properties upon recognition of target nucleic acids offer powerful tools to DNA and RNA visualization technologies. Herein, we describe a rapid and effective visualization method of nucleic acids in both fixed and living cells with hybridization-sensitive fluorescent oligonucleotide probes. These probes are efficiently quenched in an aqueous environment due to the homodimeric, excitonic interactions between fluorophores but become highly fluorescent upon hybridization to DNA or RNA with complementary sequences. The fast hybridization kinetics and quick fluorescence activation of...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Time-Lapse Imaging of Nuclear Bodies
Fluorescence microscopy is a powerful technique that has become central in the study of the structure and function of biological specimens. This is due in large part to its specificity and versatility. Although an understanding of structure—typically through high-resolution imaging of fixed material—has proved an important tool to understanding function, fluorescence microscopy also offers a mechanism to interrogate cells in the living state, providing a means to explore dynamic process within the specimen over long time periods at high temporal resolution. The cell nucleus is a highly compartmented environment...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

High-Resolution 3D DNA FISH Using Plasmid Probes and Computational Correction of Optical Aberrations to Study Chromatin Structure at the Sub-megabase Scale
We describe probe design and generation and the 3D DNA FISH procedure. We further discuss how to optimize microscope settings and to implement calibration-bead-assisted computational corrections in order to achieve 50 nm resolution in two-color distance measurements between probes that can be as close as 50 kb along the genome. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Super-Resolution Imaging of Nuclear Bodies by STED Microscopy
The sizes of nuclear bodies and other nuclear structures are normally no more than a few hundred nanometers. This size is below the resolution limit of light microscopy and thus requires electron microscopy for direct observation. Recent developments in super-resolution microscopy have extended the resolution of light microscopy to beyond 100 nm. Here, we describe a super-resolution technique, gated STED, for the analysis of the structure of nuclear bodies, with emphasis on the sample preparation and other technical tips that are important to obtain high-quality super-resolution images. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

Visualization of lncRNA by Single-Molecule Fluorescence In Situ Hybridization
Single-molecule RNA fluorescence in situ hybridization is a technique that holds great potential for the study of long noncoding RNA. It enables quantification and spatial resolution of single RNA molecules within cells via hybridization of multiple, labeled nucleic acid probes to a target RNA. It has recently become apparent that single-molecule RNA FISH probes targeting noncoding RNA are more prone to off-target binding yielding spurious results than when targeting mRNA. Here we present a protocol for the application of single-molecule RNA FISH to the study of noncoding RNA as well as an experimental procedure for valida...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news

ERRATUM: Transactivation of Large Ribozymes
(Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news

Assembly and Characterization of megaTALs for Hyperspecific Genome Engineering Applications
Rare-cleaving nucleases have emerged as valuable tools for creating targeted genomic modification for both therapeutic and research applications. MegaTALs are novel monomeric nucleases composed of a site-specific meganuclease cleavage head with additional affinity and specificity provided by a TAL effector DNA binding domain. This fusion product facilitates the transformation of meganucleases into hyperspecific and highly active genome engineering tools that are amenable to multiplexing and compatible with multiple cellular delivery methods. In this chapter, we describe the process of assembling a megaTAL from a meganuclea...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news

Ligation-Independent Cloning (LIC) Assembly of TALEN Genes
We describe a ligation-independent cloning (LIC) based method to allow high-throughput assembly of TALE nuclease genes at high fidelity and low effort and cost. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news

Efficient Design and Assembly of Custom TALENs Using the Golden Gate Platform
An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other s...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news

Engineering of Customized Meganucleases via In Vitro Compartmentalization and In Cellulo Optimization
LAGLIDADG homing endonucleases (also referred to as “meganucleases”) are compact DNA cleaving enzymes that specifically recognize long target sequences (approximately 20 base pairs), and thus serve as useful tools for therapeutic genome engineering. While stand-alone meganucleases are sufficiently active to introduce targeted genome modification, they can be fused to additional sequence-specific DNA binding domains in order to improve their performance in target cells. In this chapter, we describe an approach to retarget meganucleases to DNA targets of interest (such as sequences found in genes and cis regulato...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news

Genome Engineering Using Adeno-Associated Virus (AAV)
The ability to edit the genome of cell lines has provided valuable insights into biological processes and the contribution of specific mutations to disease biology. These techniques fall into two categories based on the DNA repair mechanism that is used to incorporate the genetic change. Nuclease-based technologies, such as Zinc-Finger Nucleases, TALENS, and Crispr/Cas9, rely on non-homologous end-joining (NHEJ) and homology directed repair (HDR) to generate a range of genetic modifications. Adeno-Associated Virus (AAV) utilizes homologous recombination to generate precise and predictable genetic modifications directly at ...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news