The analysis of cancer cell behavior in the primary tumor in living animals provides an opportunity to explore the process of invasion and intravasation in the complex microenvironment that is present in vivo. In this chapter, we describe the methods that we have developed for performing intr ...
Cell migration and metastasis are key features of aggressive tumors. These processes can be difficult to study, as they often occur deep within the body of a cancer patient or an experimental animal. In vitro assays are able to model some aspects of these processes, and a number of assays have been develo ...
The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm and high-powered imaging technology have been used to visualize the nuclear–cytoplasmic dynamics of cancer-cell in vivo. Nuclear–cytoplasmic dynamics have been imaged in cancer cel ...
RNA interference (RNAi) has rapidly become a powerful tool for drug-target discovery and therapeutics. Cancer is an important application for RNAi therapeutics, since abnormal gene regulation is thought to contribute to the pathogenesis and maintenance of the metastatic phenoty ...
Although fluorescent proteins are ubiquitously used as genetic tracers and imaging agents, there is significant room for improvement. This chapter discusses how new improved fluorescent proteins can be designed. It focuses on the design of far-red and infrared fluorescent protein ...
The discovery of multicolored fluorescent proteins (FPs), in reef corals, that are close relatives of the green fluorescent protein (GFP) has led to what is now viewed as the second GFP revolution. Numerous GFP-type proteins, termed “reef FPs,” have been cloned from reef organisms and many posse ...
Live confocal microscopy of vital fluorescent markers, expressed in mouse embryonic tissues, is a powerful and exciting method to study mammalian embryonic development. This chapter discusses imaging approaches to visualize and characterize dynamic changes of the yolk-sac vas ...
Target-specific imaging probes represent a promising tool in the molecular imaging of human cancer. Fluorescently-labeled target-specific probes are useful in imaging cancers because of their ability to bind a target receptor with high sensitivity and specificity. The develop ...
Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. The rat, as with the mouse, has offered important animal models for biology and medical research, and has provided a wealth of physiological and pharma ...
The polymerase chain reaction (PCR) is a versatile, widely used method for the production of a very large number of copies of a specific DNA molecule (1,2). For some applications, it is advantageous to subclone the PCR product into a plasmid vector for subsequent replication in bacteria (3–6). Subcl ...
Among the many variations of polymerase chain reaction (PCR)-based mutagenesis procedures, the “megaprimer” method (1–5) is probably the simplest and most versatile. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cl ...
PCR amplification has become an extremely powerful and universally used technique for cloning and manipulating DNA segments. It is especially useful for the ability to alter the terminal sequences of an amplified product simply by using primers containing the desired changes. Howeve ...
Oligonucleotide-directed mutagenesis techniques are extensively used for studying gene regulation and DNA and protein structure/function relationships. A number of PCR-based mutagenesis methods have been developed recently (1–8). In general, these PCR-based approach ...
Flip-PCR was designed to allow the sequence inversion of small regions of DNA within a regulatory region in order to examine the importance of cis-acting elements on transcriptional activity (1). Flip-PCR, a modification of the two-step overlap extension method of site-specific mutagen ...
A number of mutagenesis methods allow the systematic survey of a region of transcriptional regulatory sequence for identifying functional elements. In these methods, clusters or blocks of point mutations are introduced at discrete locations that span a suspected regulatory region ...
PCR methodology is one of the fastest available procedures for site-directed mutagenesis (1,2). However, it has been criticized for a lack of reliability because of unwanted mismatches produced during the PCR reaction (3,4). In the present protocol, we describe an improvement on the effici ...
The examination of gene expression and the search for novel and/or tissue-specific genes has been facilitated by the presence of conserved domains among the members of gene families (e.g., ref. 1). As discussed in the previous chapters of this section, such a conserved domain can be exploited by des ...
Multigene families throw a particular twist into the problem of polymerase chain reaction (PCR) primer design. One frequently needs primers that will not only amplify the sequence of interest, but at the same time fail to amplify the set of very similar sequences that comprises the remaining me ...
The use of degenerate primers in the polymerase chain reaction (PCR) is an effective method for identifying related genes that share limited sequence similarity. Other methods, using oligonucleotide or heterologous probes, for example, may fail to identify genes that are not highly con ...
The invention of polymerase chain reaction (PCR) and its application to amplification of reverse transcribed cDNA copies of mRNA has opened new possibilities for the development of techniques for identification of changes in gene expression patterns. Two very similar variants of the s ...
