General


Blood feeding mosquitoes transmit many of the world’s deadliest diseases, which are resurgent in developing countries and pose threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for disease control through the genetic manipulation of vector insects. To accomplish this, vector insects must be established as laboratory model organisms, allowing for a better understanding of their biology, and in particular, the genes that regulate their development. Aedes aegypti is a vector mosquito of great medical importance because it is responsible for the transmission of dengue fever and yellow fever. In the October issue of Cold Spring Harbor Protocols, Molly Duman-Scheel and colleagues present an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Protocols are provided for culturing and egg collection, fixation and tissue preparation, whole mount in situ hybridization, immunohistochemical analysis and RNA interference in Ae. aegypti. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

This article series marks the latest entrant in Cold Spring Harbor Protocols’ long-running series on Emerging Model Organisms.

While post-transcriptional modifications are a characteristic feature of noncoding RNAs, the biological function of these modifications is unknown. Cytosine-5 methylation has been detected in abundant RNA molecules including ribosomal RNAs and transfer RNAs, but the methylation status of cytosines in other noncoding RNAs is not known. To further investigate these modifications, Matthias Schaefer and colleagues from the German Cancer Research Center have developed a protocol for Detection of Cytosine Methylation in RNA Using Bisulfite Sequencing. The method, featured in the October issue of Cold Spring Harbor Protocols, uses a bisulfite treatment of RNA to chemically deaminate nonmethylated cytosines to uracil, leaving methylated cytosines unaffected. Subsequent cDNA synthesis and PCR amplification offers researchers material for high-throughput sequencing analysis of the methylation patterns in any RNA molecule, including noncoding RNAs and low-abundance RNAs. As one of October’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

If you’ve visited Cold Spring Harbor Protocols in the last 12 hours or so, you may have noticed that things look a little different. Welcome to Phase 1 of our re-design.

CSH Protocols was originally designed as a database, but over time, our readers and authors made it clear to us that their needs would be better served if it became more journal-like. For an author, publishing a peer-reviewed, PubMed indexed paper offers better rewards than contributing to a database. For readers who are used to digging information out of the published literature, a journal offers better findability. And so, we’ve redesigned the site to bring it into line with Cold Spring Harbor Laboratory Press’ other journal offerings.

The site still offers the same functionality, but now the navigation is greatly improved. The new design also allows us to start experimenting with widgets, adding further functionality. The next step will be migrating to our host, Highwire Press’ new H2O platform, which will allow for even further functionality to be built in (coming in the near future).

So take a look around, maybe you’ll find some things you haven’t found before. And stay tuned for future developments.

A cell devotes a significant amount of effort to maintaining the stability of its genome, preventing the sorts of chromosomal rearrangements characteristic of many cancers. Assays that measure the rate of gross chromosomal rearrangements (GCRs) are needed in order to understand the individual genes and the different pathways that suppress genomic instability. In the September issue of Cold Spring Harbor Protocols, Richard Kolodner and colleagues from the University of California, San Diego’s Ludwig Institute for Cancer Research present Determination of Gross Chromosomal Rearrangement Rates, a genetic assay to quantitatively measure the rate at which GCRs occur in yeast cells. The assay measures the rate of simultaneous inactivation of two markers placed on a nonessential end of a yeast chromosome. This simple protocol for determining GCR mutation rates in a variety of genetic backgrounds coupled with a diversity of modified GCR assays has provided tremendous insight into the large numbers of pathways that suppress genomic instability in yeast and appear to be relevant to cancer suppression pathways in humans. As one of September’s featured articles, the full text protocol is freely available to subscribers and nonsubscribers alike.

The Drosophila neuromuscular junction (NMJ) provides a superb model system for investigating the cellular and molecular mechanisms of synaptic transmission. The NMJ is large, easily accessed and its genetics are well-characterized. It shares many structural and functional similarities to synapses in other animals, including humans. In the September issue of Cold Spring Harbor Protocols, Bing Zhang and Bryan Stewart present an essential set of primers for electrophysiological recording from the Drosophila NMJ. The issue contains a detailed explanation of the Equipment Setup necessary, as well as instructions for Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes. Protocols for Electrophysiological Recording from a ‘Model’ Cell, Electrophysiological Recording from Drosophila Larval Body-Wall Muscles, Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction, and Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Neuromuscular Junction are also included. These protocols are adapted from Drosophila Neurobiology: A Laboratory Manual. Based on Cold Spring Harbor Laboratory’s long-running course, this manual has rapidly become an important resource for any neuroscience lab.

Producing recombinant proteins in bacterial hosts is a widely-used laboratory procedure. But generating a large yield of protein is often challenging. Getting enough raw material for experiments can be a time-consuming and frustrating process. In the August issue of Cold Spring Harbor Protocols, Jianjun Wang and colleagues present a method for Preparation of Very-High-Yield Recombinant Proteins using Novel High-Cell-Density Bacterial Expression Methods. By combining traditional IPTG induction with high-cell-density auto-induction, the method routinely produces 15-35 mg of pure protein from 50 mL bacterial cell cultures. Detailed protocols are given for preparation of a starting culture, double colony selection and optimization of expression conditions, which ensure plasmid stability resulting in a high yield of recombinant protein production.

While it is possible to analyze the global lipid composition of a cell, a deeper understanding of what lipids are doing within that cell is more difficult to come by. Though the lipid components may be known, finding their exact position, how dynamically they change location, and how rapidly they are metabolized presents an experimental challenge. The obvious approach would be the addition a fluorescent tag, which would allow for imaging of lipids in cells. Unfortunately, most commonly used fluorescent tags are as large as the lipid itself and are likely to have a strong effect on lipid location and metabolism.

In the July issue of Cold Spring Harbor Protocols, Joachim Goedhart and colleagues present a suite of protocols to get around these problems and allow for live imaging of lipids in cells. Their introduction to the topic explains the approach:

To circumvent this problem, two solutions have been developed–namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell.

Protocols are provided for Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain, Labeling Lipids for Imaging in Fixed Cells, and Labeling Lipids for Imaging in Live Cells.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

Cold Spring Harbor Laboratory Press’ new Drosophila Neurobiology laboratory manual covers the three main approaches taught in the CSHL course: studying neural development, recording and imaging the nervous system, and studying behavior. The featured electrophysiology paper is part of the recording/imaging section, while the second featured article in the July issue of Cold Spring Harbor Protocols comes from a neural development chapter.

The larval Drosophila brain has been a valuable model for investigating the role of stem cells in development. These neural stem cells, called “neuroblasts,” have provided insight into the role of cell polarity in influencing cell fate. Identifying neuroblasts and their progeny requires a method capable of recognizing cell polarity and cell fate markers. Immunofluorescent Staining of Drosophila Larval Brain Tissue, provided by Cheng-Yu Lee and colleagues, describes procedures for the collection and processing of Drosophila larval brains for analysis of these markers. Neuroblasts are identified via immunolocalization, the use of labeled antibodies that specifically bind the marker proteins of interest. As one of our featured articles, it is freely available to subscribers and non-subscribers alike.

The June issue of Cold Spring Harbor Protocols includes an early preview of CSHL Press’ forthcoming RNA: A Laboratory Manual. Protocols covering basic RNA techniques are now available, including methods for purification of RNA by by SDS Solubilization and Phenol Extraction and by Using TRIzol (TRI Reagent), Ethanol Precipitation of RNA and the Use of Carriers, Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells, Removal of Ribosomal Subunits (and rRNA) from Cytoplasmic Extracts before Solubilization with SDS and Deproteinization, Removal of DNA from RNA, Nondenaturing Agarose Gel Electrophoresis of RNA and Polyacrylamide Gel Electrophoresis of RNA.

The last two on that list cover gel electrophoresis, two of the most important and frequently used techniques in RNA analysis. Electrophoresis is used for RNA detection, quantification, purification by size and quality assessment. Gels are involved in a wide variety of methods including northern blotting, primer extension, footprinting and analyzing processing reactions. The two most common types of gels are polyacrylamide and agarose. Polyacrylamide gels are used in most applications and are appropriate for RNAs smaller than approximately 600 nucleotides (agarose gels are preferred for larger RNAs). Polyacrylamide Gel Electrophoresis of RNA describes how to prepare, load and run polyacrylamide gels for RNA analysis. The is featured in the June issue, and as one of our featured articles, the full-text version is available to subscribers and non-subscribers alike.

This set is just a small sampling of the manual’s contents, basic techniques from an early chapter. The full table of contents can be seen here.

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