The diverse array of applications benefiting from fluorescent proteins ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. Fluorescent proteins have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In the December issue of Cold Spring Harbor Protocols, David Piston and colleagues present Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants, a comprehensive overview of the wide variety of fluorescent proteins that are currently available. The article features more than twenty movies of different fluorescent proteins in action and is a great primer for planning imaging experiments. As one of December’s featured articles, it is freely available to subscribers and non-subscribers alike.

In addition, the same authors have also contributed Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of fluorescent proteins and imaging of several fluorescent proteins at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from flourescent proteins.

Both articles are adapted from the spectacular new manual, Live Cell Imaging: A Laboratory Manual, Second Edition which is due out by month’s end.

CSH Protocols December Cover

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

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.

Quick Miniprep for Plant DNA Isolation gives a rapid method, good when processing large numbers of samples where high purity of the resulting DNA is not needed.

Dellaporta Miniprep for Plant DNA Isolation is quick and inexpensive and results in a higher quality end product. Because it yields enough DNA to test a large number of markers, it’s recommended in cases when many markers will be tested on the same samples.

Cetyltrimethyl Ammonium Bromide (CTAB) DNA Miniprep for Plant DNA Isolation is useful for isolation of DNA from tissues containing high amounts of polysaccharides. The presence of polysaccharides can inhibit PCR reactions. Under high salt conditions, CTAB binds the polysaccharides and takes them out of solution.

These articles join a previously published protocol for plant DNA purification, from C. Eduardo Vallejos, An Expedient and Versatile Protocol for Extracting High-Quality DNA from Plant Leaves. This method results in DNA that has virtually no protein or phenolic contaminants, is RNA-free, contains high-molecular-weight fragments according to CHEF electrophoresis, and is fully digestible by restriction enzymes.

The moss Physcomitrella patens has been used in laboratory research for more than 80 years, but the last 15 have seen a resurgence in moss research. P. patens can easily be grown in the lab, and spends most of its life in a haploid state that allows many of the approaches used in yeast and microbes to be applied. Methods for RNAi have been worked out, the genome has been sequenced and assembled, physical and genetic maps are available, and more than 250,000 expressed sequence tags (ESTs) are known. Ralph Quatrano and colleagues have contributed an overview of the use of P. patens as a laboratory organism to February’s issue of Cold Spring Harbor Protocols along with protocols for culture, isolation of protoplasts, somatic hybridization, chemical and UV mutagenesis, transformation via direct DNA uptake, T-DNA mutagenesis, and biolistic delivery systems, and isolation of DNA, RNA and proteins.

Choanoflagellates are a varied group of protozoa that are the closest living relative to the metazoa, and their study is leading to new insights into metazoan ancestry and origins. Barry Leadbeater and colleagues have written up a series of articles highlighting the use of Monosiga brevicollis as a representative species, as it has recently had its genome sequenced, and is readily grown and manipulated in the laboratory (protocols included are generally transferable to most choanoflagellate species). The set of articles includes an overview of choanoflagellates, and protocols for isolation from field samples and culture of choanoflagellates, long-term storage, visualization of actin and beta-tubulin, purification of total RNA, and preps for rapid DNA isolation, high molecular weight DNA isolation and separation of choanoflagellate and bacterial genomic DNA.

These articles on Emerging Model Organisms are being collected in a series of lab manuals, the first of which is currently available here (now on sale 25% off!).

Like all of our featured articles, access to this protocol is free for both subscribers and non-subscribers alike.

Much of twentieth century biological research has focused on a limited number of model organisms, such as Arabidopsis, C. elegans, mouse, Drosophila, and E. coli. These classical model species, chosen because they are amenable to laboratory research and suitable for studying a range of biological problems, have served to elucidate many biological processes that can be generalized across a wider array of organisms. It is only a slight exaggeration to say that the basic workings of the cell were elucidated mostly from experiments on a few single-celled organisms — primarily E.coli and yeast. Our understanding of animal development was largely based on the genetics of fruit fly and worm and on the manipulation of a handful of amphibians and mouse; most of what we learned about the molecular and developmental biology of plants came from examining Arabidopsis and just a few other species. But biology wasn’t always done this way.
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As an example, early efforts to understand gastrulation were informed by careful analysis of a wide variety of organisms (a superb summary of this research can be found in Scott Gilbert’s chapter in Gastrulation). Ernst Haeckel’s Gastrea theory, as he openly acknowledged, relied heavily on the exhaustive work of Alexander Onufreevich Kowalevsky, who described early embryonic development in Amphioxus, Phallusia, Ascidia, Phoronix, Echinus, Ophiura, Limnaeus, the frog, comb jellies, sturgeons and Petromyzon. Modern biologists, by contrast, have only rarely strayed from the relatively small handful of established model organisms. Studies in these models have resulted in a great depth of knowledge, as the concentration of many on a limited number of systems has proved a valuable approach. But things are changing again; with the evolution of new technologies, new questions and new approaches are now possible.

The time and costs of sequencing genomes continues to drop. Techniques for selectively altering the expression patterns of genes have become more generally applicable across species. And more and more biologists are expanding their interests from the purely mechanistic to embrace evolutionary considerations. Because of these factors, we are now seeing a great expansion in the variety of organisms regularly studied. Researchers are now introducing new species to the laboratory, opening up new avenues of research and allowing comparison and refinement of our understanding of already-established models.

October sees the publication of a new article type from CSH Protocols, the “Emerging Model Organism“. The goal of this new venture is to introduce researchers to the new generation of model organisms, and to provide a diverse catalog of potential species useful for extending research in new directions. Each article presents a new organism (or group of related organisms) and provides a detailed explanation of why they are useful for laboratory research, along with information on husbandry, genetics and genomics, and pointers towards further resources. Most are accompanied by a set of basic laboratory protocols for working with that organism. This month brings the introductory articles in this series, covering the nematode Pristionchus pacificus, The American Wandering Spider Cupiennius salei, and the Opossum Monodelphis domestica. Our featured articles for the month, freely available to subscribers and non-subscribers are:
The Genus Antirrhinum (Snapdragon): A Flowering Plant Model for Evolution and Development
and
Planarians: A Versatile and Powerful Model System for Molecular Studies of Regeneration, Adult Stem Cell Regulation, Aging, and Behavior

The first set of articles in this series is being collected and will be available in November as a printed laboratory manual. We hope that these Emerging Model Organism articles will serve as a practical guide for finding just the right species for addressing your research needs, and as a primer for introducing new systems to your laboratory.

This series would not have been possible without the efforts of a special editorial board assembled for this project, Richard Behringer, Alexander Johnson, Robb Krumlauf, Mike Levine, Nipam Patel and Neelima Sinha. They have done a superb job of honing both the format of the content of these articles, and in pointing us toward leading experts and valuable new model systems. We also welcome your suggestions for organisms to include in future volumes of this series – please send us your ideas by contacting us at submitprotocols@cshl.edu.