Fluorescent protein methods: strategies and applications.
Fluorescent proteins such as the "green fluorescent protein" (GFP) are popular tools in Caenorhabditis elegans, because as genetically encoded markers they are easy to introduce. Furthermore, they can be used in a living animal without the need for extensive sample preparation, because C. elegans is transparent and small enough so that entire animals can be imaged directly. Consequently, fluorescent proteins have emerged as the method of choice to study gene expression in C. elegans and reporter constructs for thousands of genes are currently available. When fused to a protein of interest, fluorescent proteins allow the imaging of its subcellular localization in vivo, offering a powerful alternative to antibody staining techniques. Fluorescent proteins can be employed to label cellular and subcellular structures and as indicators for cell physiological parameters like calcium concentration. Genetic screens relying on fluorescent proteins to visualize anatomical structures and recent progress in automation techniques have tremendously expanded their potential uses. This chapter presents tools and techniques related to the use of fluorescent proteins, discusses their advantages and shortcomings, and provides practical considerations for various applications.
Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada.
Harald
H
Hutter
D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*; D019076:Transgenes
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins