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.

Metagenomics, the study of DNA isolated from naturally occurring populations and samples, is rapidly growing. Improvements to cloning and sequencing techniques are allowing researchers to study organism in environmental samples, and new knowledge of species interactions and community dynamics is emerging. The identification of microorganisms in these samples is of vital importance to their interpretation. In the January issue of Cold Spring Harbor Protocols, Annelie Wendeberg of the Helmholtz Centre for Environmental Research presents a protocol for Fluorescence In Situ Hybridization for the Identification of Environmental Microbes. The methods described allow the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes followed by signal amplification with catalyzed reporter deposition (CARD). The protocol is one of January’s featured articles, and like all featured articles in Cold Spring Harbor Protocols, it is freely accessible to subscribers and non-subscribers alike.

Cold Spring Harbor Protocols is hosting the movie figures that accompany the new lab manual, Live Cell Imaging, Second Edition, edited by Robert Goldman, Jason Swedlow and David Spector, . These movies are freely accessible to all, and worth a look if you’re interested in seeing the state of the art in time lapse imaging.

We’re getting toward the end of the second volume of our Emerging Model Organisms series in Cold Spring Harbor Protocols, and November’s issue brings us a look at the Hawaiian Bobtail Squid and the genus Dioscorea, or True Yams.

Euprymna scolopes, the Hawaiian Bobtail Squid (our cover model this month, see below) is a cephalopod that’s well-suited for study in the laboratory. E. scolopes is primarily studied in three contexts:
1) as a model for cephalopod development–the embryos and protective chorions are clear, making it amenable for the observations and manipulations common in other studied model systems
2) as a model of animal-bacteria symbioses with the luminous marine bacterium Vibrio fischeri
3) as a system for studying the interaction of tissues with light, as the squid features a specialized light organ.

Heinz Gert de Couet and colleagues supply an overview of the Hawaiian Bobtail Squid as a model system, along with protocols for Preparation of Genomic DNA, Confocal Immunocytochemistry, Whole-Mount In Situ Hybridization (parts 1 and 2), and Culture and Observation.

Dioscorea is a large genus of plants that are monocots but that look like dicots, and are closely related to the phylogenetically derived group containing the grasses. It’s interesting evolutionarily because of the position it occupies, as a link between the eudicots and grasses–groups that contain all the model flowering plant species. The true yam is also important as a food crop. R. Geeta and colleagues provide an overview of the genus, and protocols for husbandry, culturing tissues, management of plantlets, controlled crosses, and DNA extraction.

CSH Protocols November Cover

Volume 2 of our Emerging Model Organisms series rolls on in the October issue of Cold Spring Harbor Protocols. This month brings a look at two emerging models, one all-time classic.

Neelima Sinha and colleagues present “The Mother of Thousands” (Kalanchoë daigremontiana), a plant which has the fascinating ability to regenerate and entire organism from somatic cells. The process of forming a somatic embryo outside of a seed environment provides an attractive model system for studying embryogenesis. Kalanchoë is also used in the study of Crassulacean acid metabolism (CAM), which is an important evolutionary adaptation of the photosynthetic carbon assimilation pathway to arid environments. In addition, natural compounds extracted from tissues of Kalanchoë have potential applicability in treating tumors and inflammatory and allergic diseases, and have been shown to have insecticidal properties. Protocols are provided for fixing and sectioning tissues, in situ hybridization, transformation using agrobacterium, DNA extraction and RNA extraction.

John Werren and colleagues provide The Parasitoid Wasp Nasonia: An Emerging Model System with Haploid Male Genetics. Nasonia is a genus consisting of four interfertile species. They’re particularly useful as a genetic tool for study because females are diploid and develop from fertilized eggs, and males are haploid and develop from unfertilized eggs. This allows geneticists to exploit many of the advantages of haploid genetics in an otherwise complex eukaryotic organism. Protocols are available for field collection, strain maintenance, rearing fly hosts, egg collection, virgin collection and crossing methods, larval RNAi and curing Wolbachia bacterial infections.

As for that “classic” system mentioned above, if you know genetics, then you know Barbara McClintock, and you know that Maize has been a keystone model system for nearly a century. Micheal Scanlon and colleagues have written up Maize (Zea mays): A Model Organism for Basic and Applied Research in Plant Biology, which gives an up-to-date discussion of the state of Maize research.

RNA molecules interact with proteins to drive many cellular activities, including post-transcriptional processing of RNA, regulation of translation, and transport of RNA to name but a few. These ribonucleoprotein complexes are isolated by coimmunoprecipitation (co-IP), where a protein-specific antibody is used to purify the protein of choice and its associated complex members. Analysis of RNA-Protein Complexes by RNA Coimmunoprecipitation and RT-PCR Analysis from Caenorhabditis elegans gives step-by-step instructions for RNA co-IP from C. elegans whole-worm extracts. The protocol, from Christian Eckmann and colleagues at the Max Planck Institute of Molecular Cell Biology and Genetics, starts with the large-scale growth of worms and describes the preparation of whole-worm extracts, RNA co-IP, isolation of the purified RNA, and identification of specific genes through RT-PCR. As one of our featured articles for October, the protocol is freely available to subscribers and non-subscribers alike. Eckmann and colleagues have also contributed an accompanying article describing Analysis of In Vivo Protein Complexes by Coimmunoprecipitation from Caenorhabditis elegans.

The study of RNA has long been the tool of choice for understanding where and when genes are expressed in a cell, tissue, or organism during development or under specific physiological or environmental conditions. Recent discoveries have revolutionized our concept of RNA function; it is now known to be active in a much wider set of biological processes than was previously believed. Techniques for isolating RNA and for uncovering its interactions with proteins have taken on new importance as many laboratories define the roles of specific RNAs in the cell. The October issue of Cold Spring Harbor Protocols features articles detailing methods for RNA analysis.

Quantitative Real-Time RT-PCR (qRT-PCR) of Zebrafish Transcripts: Optimization of RNA Extraction, Quality Control Considerations and Data Analysis from Donald Love and colleagues at the University of Auckland presents an optimized method for RNA isolation from zebrafish, along with quality assessment and the use of reference genes. A protocol for quantitative real-time polymerase chain reaction (qRT-PCR) is also included. Like all of our featured articles, these protocols are freely available to subscribers and non-subscribers alike.

Our long-running series of articles highlighting emerging model organisms continues in September with three entries, The Starlet Sea Anemone (Nematostella vectensis), Cephalochordates (Amphioxus or Lancelets) and The Western Clawed Frog (Xenopus tropicalis).

The slow rate of sequence evolution, the presumed high degree of preservation of ancestral traits, the ease of culturing, and the availability and experimental tractability of the early embryos have made Nematostella a prime cnidarian model for a number of biological studies. It serves not only as a model system for cnidarians, but also as an important representative of its phylum in comparisons with other lower Metazoa or Bilateria. Ulrich Technau and colleagues provide an overview of Nematostella, and protocols for spawning, in situ hybridization, antibody and phalloidin staining and BrdU labeling.

Cephalochordates, commonly called amphioxus or lancelets, are marine invertebrate chordates. Studies on cephalochordates have answered some long-standing questions concerning the evolution of vertebrates from their invertebrate ancestors and have also generated interesting avenues for further investigation of the evolutionary origin of developmental mechanisms that led to the emergence of the vertebrate body plan. Linda Holland and colleagues provide background on Cephalochordates, along with detailed methods for Amphioxus embryo collection, in situ hybridization, DNA extraction, and RNA extraction and extracting RNA from small amounts of tissue for RT-PCR.

Xenopus tropicalis is a small, wholly aquatic frog that is a diploid relative of Xenopus laevis. It shares many of the advantages of X. laevis as a model organism for studying aspects of vertebrate biology, particularly the genetic, biochemical, and environmental factors that influence vertebrate development from embryonic stages through adulthood. X. tropicalis is also finding uses as an important test species for assessing the impact of environmental toxins and disease on amphibians, which are in decline in many areas of the world due to water-borne pollutants and infectious agents such as the chytrid fungus. Frank Conlon and colleagues have contributed an overview of X. tropicalis, along with protocols for natural mating, in vitro fertilization, and tissue sampling and genomic DNA preparation.