The May issue of CSH Protocols is now live, and in it you’ll find featured articles on classic techniques for the analysis of chromosomes. With the leaps and bounds being made in epigenetics these days, knowing your way around chromatin becomes even more valuable. This month’s freely accessible articles give you methods for chromosome analysis in Drosophila and in Mouse.

Mapping Protein Distributions on Polytene Chromosomes by Immunostaining takes advantage of the formidable size and structure of the large polytene chromosomes found in Drosophila salivary glands. These easily dissected chromosomes allow mapping of chromosomal protein distributions at very high resolution. May’s issue also contains a protocol for the Dissection of Larval Salivary Glands and Polytene Chromosome Preparation.

The second featured method for May, Karyotyping Mouse Cells, is drawn from the widely used laboratory manual Manipulating the Mouse Embryo. A karyotype is a visual presentation of a cell’s chromosomes, and can be used as a test for quickly identifying chromosomal abnormalities.

April’s issue of CSH Protocols features a set of articles on the production and use of retroviral vectors for gene transfer from Kenneth Cornetta, Karen Pollok and Dusty Miller. Retroviral Vectors for Gene Transfer provides an overview of the subject, drawing on the more than twenty years of experience researchers have with the use of these vectors. The advantages of retroviral vectors are detailed (efficiency, integration and ease of production) along with the disadvantages (inactivation, a requirement for cell division and possible oncogenic activation). The authors discuss important aspects of vector design and choice of packaging cell lines.

Four protocols are provided, two for production of viral vectors, and two for their use in transducing cells. Detailed methods are offered for Retroviral Vector Production by Transient Transfection, and for the Generation of Stable Vector-Producing Cells. Once vectors are generated, they can easily be used to Transduce Cell Lines which are actively proliferating. However, using retroviral vectors with primitive progenitor or stem cells, which are not continuously dividing, is much less efficient. In Transduction of Primary Hematopoietic Cells by Retroviral Vectors, the authors describe two interventions to improve efficiency of transfer, the use of cytokines and other growth factors to stimulate cell cycling, and the use of matrix proteins to mediate colocalization of target cells and vector.

The March issue of CSH Protocols includes a diverse trio of methods for fluorescently labeling cells and subcellular structures. The most basic of the three methods comes from Brad Chazotte at Campbell University and covers labeling of lysozymes with Neutral Red. This joins a group of already-published protocols from Chazotte on labeling cellular structures including the plasma membrane, the golgi apparatus and acetylcholine receptors. Expect more articles in this series in forthcoming issues.

The second protocol provides a method for differentiating viable plant cells from dead plant cells. Contributed by Birgit Schwab and Martin Hülskamp from the Center for Plant Molecular Biology in Tübingen, the technique takes advantage of the inability of propidium iodide to enter live cells. Dead cells allow it in, and fluoresce red, so they can be easily identified.

Finally, Paul Kulesa’s group at the Stowers Institute have written up their method for Photoactivation Cell Labeling for Cell Tracing in Avian Development. This technique allows for selective marking of individual cells or groups of cells at precise times and spatial locations normally not accessible using previous techniques. It’s less invasive than most methods used for labeling cells in avian embryos, and can be targeted to both individual cells, or small groups of cells. This month’s cover image shows an example of this technique.

The March issue of CSH Protocols has two featured (freely available) protocols on high-throughput methods for studying gene regulation.

The first method approaches regulatory analysis through epigenetic mechanisms. Methylated CpG Island Amplification and Microarray (MCAM) for High-Throughput Analysis of DNA Methylation, developed by Marcos Estecio and Jean-Pierre Issa of the MD Anderson Cancer Center, and Pearlly Yan and Tim Huang of the Ohio State University Comprehensive Cancer Center is a rapid, genome-wide method for identifying regions where methylation is occurring. This protocol has proven successful for proven successful for use in comparing normal tissues and tumors, helping researchers better understand the factors responsible for cancer.

The second protocol looks at the binding of regulatory proteins to DNA and their role in transcriptional regulation. The method, DNA Immunoprecipitation (DIP) for the Determination of DNA-binding Specificity, allows researchers to determine the specific DNA sequence that a regulatory protein binds. The technique allows for rapid screening of the entire genome for these binding sites, which gives insight into which genes these protein factors control.

Immunohistochemistry (the localization of proteins in a tissue by binding antibodies to specific antigens) is a technique where one protocol definitely does not fit all. Each model organism seems to have its own quirks, whether it be in the fixative used, the methods needed for antibody penetration, issues with autofluorescence or even just figuring out which cross-species antibodies work in a given system. To that end, we’ve been working on expanding our coverage of immunohistological protocols. The February issue of CSH Protocols brings methods for plant sections, using both avidin-biotin and alk-phos, as well as a method for whole-mount immunocytochemistry in Xenopus embryos from John Wallingford’s lab at the University of Texas (they provided the lovely cover image for this month).

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Those familiar with the CSHL Press Manual, Drosophila Protocols (edited by Sullivan, Ashburner and Hawley) will want to be sure to check CSH Protocols’ February issue, as Bruce Paterson’s group at the National Institutes of Health has written an update of his chapter, “Targeted Disruption of Gene Function in Drosophila by RNA Interference”. The new, up-to-date version of the book chapter appears online in a series of articles, including Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi), Collection of Drosophila Embryos for RNA Interference (RNAi), and Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi) (freely available as one of this month’s featured protocols). Paterson’s group has also contributed a new article covering Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector.

The January Issue of CSH Protocols features several articles detailing the use of nanoparticles for gene delivery. Drug delivery methods using nanoparticles have revolutionized the field. The traditional methods for drug delivery, via oral and intravenous routes, are inefficient, non-specific and expensive. Nanoparticles allow for much greater control over delivery, targeting to specific tissues, higher stability (which allows lower doses to be used) and they can be manufactured cheaply in large quantities. Nanoparticles made from natural polymers are preferred over synthetic ones because of their greater biocompatibility and biodegradibility.

These advances in therapeutic drug delivery techniques also bring benefits to researchers at the laboratory bench. Just as nanoparticles can be used for drug delivery, they can also be used for DNA delivery. Once inside the cell, the key to efficient transfection is getting the DNA through the nuclear membrane. Mansoor Amiji’s group at Northeastern University contribute a series of articles on the use of gelatin nanoparticles for gene delivery, including a general overview, preparation and loading of gelatin nanoparticles, studying intracellular trafficking using TEM and gold-encapsulated nanoparticles, and analysis of transfection using fluorescence microscopy and FACS. In the same issue, you’ll find a protocol for preparation and transfection using biodegradable nanoparticles made from biocompatible polymers such as poly(D,L-lactide-co-glycolide) (PLGA) or polylactide (PLA) from Vinod Labhasetwar’s group at the University of Nebraska.

You can also find several related articles in previous issues of CSH Protocols, including Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery, Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery and An Overview of Condensing and Noncondensing Polymeric Systems for Gene Delivery.

January’s issue of CSH Protocols is now available online, and it contains a set of protocols from Cathy Krull’s lab at the University of Michigan. The articles provide methods for electroporating your gene of interest into somites, neural crest cells and motor neurons. The accessibility of the chick embryo has long made it a standard model organism for developmental biology, and methods like these greatly enhance our abilities to tag and track cells, as well as to genetically manipulate the embryo. They’re even valuable for labs not working with avian systems, particularly mouse labs, because they offer the opportunity to get a quick and easy look at expression and potential effects of experimental constructs. Unlike making a transgenic mouse, an expensive and time-consuming process, working with chick eggs is inexpensive, and relatively rapid. Testing your mouse constructs in the chick embryo is a great way to fine tune the constructs themselves to ensure proper expression. It can also give insight into potential effects of construct expression, which can save valuable time once your transgenic mice are available, as you may already know where to start analyzing.

Fate-mapping, the tagging of specific cells or tissues in an embryo, and following their movements and development over time, has a long history as a valuable method. The earliest fate-maps date back to the 1880’s. The first “modern” fate-maps were created in 1929 by Walter Vogt, who applied vital dyes to regions of the amphibian embryo. This allowed him to track which embryonic regions developed into which adult tissues. Two methods, featured in the December issue of CSH Protocols and freely available to non-subscribers, present new fate-mapping techniques, which overcome some serious experimental barriers.
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In situ hybridization of mRNA has long been a standard laboratory practice. Over recent years, the technique has evolved from the laborious sectioning of tissues and treatment with radioactive probes to the easier colorimetric (and occasionally fluorescent) methods now in use. Recent issues of CSH Protocols featured articles detailing in situ hybridization in Xenopus, Drosophila (here as well), and cultured cells.

November’s issue of CSH Protocols provides a full set of instructions for in situ hybridization on mouse embryos, as well as a cutting-edge method for imaging real-time gene expression in living systems with single-transcript resolution.

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