Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

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.

Since the early days of the 20th century and Thomas Hunt Morgan’s famous “Fly Room” at Columbia University, the fruit fly Drosophila melanogaster has been at the forefront of biological research. The powerful arsenal of experimental methods developed for this model organism is now being used to tackle one of the great scientific challenges of a new century: understanding the nervous system. Cold Spring Harbor Laboratory’s Neurobiology of Drosophila course has served as the training ground for a generation of scientists tackling these complex problems. A new laboratory manual based on the protocols and background information taught in the course promises to spread these techniques to a wider audience. Methods from the manual are featured in the July issue of Cold Spring Harbor Protocols.

When a fly is confronted with danger, it jumps into the air and flies away. The giant fiber system (GFS) of Drosophila is a neuronal circuit that mediates this escape response. The neurons in the GFS are readily identified and easily accessible for experimental assay. Electrophysiological Recordings from the Drosophila Giant Fiber System, from Marcus Allen and Tanja Godenschwege, describes a simple procedure for stimulating neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS. As one of our featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

The rapid pace of technological progress in biological imaging has provided great insight into the processes of embryonic development. But for higher organisms with opaque eggs or internal development, optical access to the embryo is limited. While various embryonic culture methods are available, vertebrate development is best studied in an intact embryo model, one in which the natural environment has not been disrupted. In the June issue of Cold Spring Harbor Protocols, Paul Kulesa and colleagues from the Stowers Institute for Medical Research present In Ovo Live Imaging of Avian Embryos, a detailed set of instructions for time-lapse imaging of fluorescently labeled cells within a living avian embryo. During the procedure, a hole is made in the shell, and a Teflon membrane that is oxygen-permeable and liquid-impermeable is used to provide a window for visualization of the embryo via confocal or two-photon microscopy. Imaging can take place for up to five days without dehydration or degradation of the normal developmental environment. As one of June’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike. Kulesa’s group also supplies a second protocol in the issue, covering Multi-Position Photoactivation and Multi-Time Acquisition for Large-Scale Cell Tracing in Avian Embryos, a technique that produced June’s cover image.

The generation of transgenic plants can be a lengthy and difficult process. Transient expression assays have been developed as faster and more convenient alternatives for investigating gene function. These assays often take advantage of the ability of Agrobacterium to transfer foreign DNA into plant cells with intact cell walls. Agrobacterium-mediated transformation is, however, inefficient and shows great variability. In the May issue of Cold Spring Harbor Protocols, Andreas Nebenführ and colleagues from the University of Tennessee present FAST Technique for Agrobacterium-Mediated Transient Gene Expression in Seedlings of Arabidopsis and Other Plant Species, a quick, efficient and economical assay for gene function in intact plants. The technique involves cocultivation of young plant seedlings and Agrobacterium in the presence of Silwet-77. The Silwet-77 facilitates transformation, thus replacing a wounding or device-dependent vacuum step. As one of May’s featured articles, it is freely available to subscribers and non-subscribers alike.

The large size and external development of the frog Xenopus laevis make it an ideal system for in vivo imaging of dynamic cellular activity. Xenopus embryos are amenable to simple genetic manipulation techniques including knockdowns and misexpression, as well as transgenesis. The ease of collecting large numbers of embryos and the larger size of individual cells within an embryo as compared with other vertebrate model systems provides an excellent platform for the observation of cellular behavior and subcellular processes. In the May issue of Cold Spring Harbor Protocols, John Wallingford and colleagues from the University of Texas provide a suite of articles detailing live imaging of Xenopus laevis at low magnification, confocal imaging of fixed tissues, and in one of May’s featured articles, High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology. This protocol describes methods for labeling and high-magnification time-lapse imaging by confocal microscopy. Like all of our featured articles, it’s freely available to subscribers and non-subscribers alike.

The incorporation of thymidine analogues, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair and other aspects of DNA metabolism. In Genome-Wide Analysis of DNA Synthesis by BrdU Immunoprecipitation on Tiling Microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae, Oscar Aparicio and colleagues from the University of Southern California couple BrdU immunoprecipitation with DNA microarrays to enable genome-wide identification of BrdU-labeled chromosomal DNA. BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression. As one of February’s featured articles in Cold Spring Harbor Protocols, the protocol is freely available to subscribers and non-subscribers alike.

mRNA in situ hybridization is a standard laboratory technique for analyzing gene expression. In a small, transparent specimen like a zebrafish embryo, this technique is straightforward and works well. Cold Spring Harbor Protocols has a set of protocols (here, here and here) describing the method from Cecilia Moens. But what happens when you’re dealing with a larger, opaque zebrafish tissue like the adult brain? Unlike mammals, zebrafish exhibit intense ongoing neurogenesis in all areas of the central nervous system. Adult zebrafish are increasingly being used in behavioral studies as well. Because the number of antibodies useful for examining expression in zebrafish is limited, mRNA in situ hybridization is a vital tool for understanding what’s happening during these processes. In the February issue of Cold Spring Harbor Protocols, Reinhard Köster and colleagues from the Helmholtz Zentrum München provide an adaptation of the standard in situ method that deals with these larger, opaque tissues by staining them after vibratome sectioning, Analysis of Gene Expression by In Situ Hybridization on Adult Zebrafish Brain Sections. While the brain is used as the sample tissue in this protocol, it can easily be modified for analysis of other adult tissues.

January’s issue of Cold Spring Harbor Protocols wraps up the second volume of our ongoing Emerging Model Organisms series. The idea behind the series is that technical advances have allowed for great expansion in the range of organisms used for research. Each set of articles is meant to introduce the reader to a new organism, to explain why it’s useful for laboratory research and to provide information on husbandry, genetics and genomics, and a set of basic laboratory protocols. The first set of 23 emerging model systems was collected in a laboratory manual, and the current set of 18 will soon be as well. January’s organisms are:

The Rabbit (Oryctolagus cuniculus): The rabbit is a valuable animal model for a variety of biomedical research areas including in vitro fertilization, early embryology and organogenesis, neurophysiology, ophthalmology, and cardiovascular research. The rabbit is also used as a model for toxicology studies and analyses of drug effects on embryo and fetal development, as well as for research involving the immune system, not to mention its common use in antibody production. Christoph Viebahn and colleagues from the University of Göttingen provide an overview of the rabbit as an experimental system, and protocols for mating and embryo isolation, dissection and fixation of embryos, embryo culture, staining and imaging, immunofluorescence, in situ hybridization, mounting, embedding and sectioning, embryo transfer, artificial insemination and cryopreservation of embryos.

Paramecium tetraurelia: Paramecium makes an interesting unicellular model, as the authors note:

Paramecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.

Eric Meyer and colleagues from the CNRS have written an overview of P tetraurelia as a model system, and protocols for maintaining cell lines, mass culture, gene silencing, DNA microinjection, immunocytochemistry, and fluorescence in situ hybridization.

We have some new organisms in the works for Volume 3, but would welcome your suggestions.