Search

The fluorescence labeling technique is a method with a high degree of specificity and sensitivity and is often chosen as a tool in the study of protein expression and subcellular compartments (1). Recently, a large number of fluorescent dyes with distinct fluorescence excitation and emission spectra have been engineered to be used in multilabeling and co-localization experiments. Some of the most common fluorescent dyes are fluorescein isothiocyanate (FITC), Cy2, Cy3, Cy5, TRITC, and rhodamine. These dyes can be excited independently using different laser wavelengths and observed in separate fluorescent channels. The efficiency of the fluorescent probes is, however, hampered by a variable degree of spectral overlap, low quantum efficiencies and extinction coefficients, or rapid photobleaching. [ABSTRACT FROM AUTHOR]

Immunocytochemistry is the most diffuse technique to visualize and localize specific biochemical components in cell compartments and tissues. With this method the antigens are tagged by antibodies that can be visualized with appropriate markers attached directly (direct method) or indirectly (indirect method) to them. The colloidal gold marker system (1) is the most widespread immunoelectron microscopy label for postembedding immunocytochemistry. Sections of embedded tissue are incubated with a primary antibody against a particular antigen, which is exposed on the surface of the section, and then with a secondary antibody conjugated to colloidal gold particles of a particular size (indirect method). It is possible to enlarge the colloidal gold size with silver enhancement. This technique is ideal for labeling both intracellular and surface antigens and it can be used for multiple labeling experiments and quantitative studies (2). [ABSTRACT FROM AUTHOR]

With this method, antibiotic selection of the infected cells gives rise to a countable number of colonies after 7-10 d (1-4). Other reporter genes are based on green fluorescent protein (GFP; ref. 5) or the Escherichia coli lacZ gene, which encodes for β-galactosidase for X-gal staining (4,6). GFP-expressing cells can be easily visualized under an ultraviolet microscope. The main advantage is that GFP expression can be detected in living cells (5). Retroviral and lentiviral transfer vectors can be engineered with an internal phosphoglycerate kinase (pgk) promoter driving the expression of a bacterial gene that confers resistance to a certain antibiotic (1, 2). Usually, the bacterial genes used are based on neomycin phosphotransferase, hygromycin B phosphotransferase, or puromycin N-acetyltransferase, which render mammalian cells resistant to G418, hygromycin B, and puromycin, respectively (1). In addition, either the internal pgk or SV40 early promoter can drive the expression of lacZ gene (4,6) or GFP (5). Functional titer does not provide a consistent measurement of virion concentration because it depends upon the transduction efficiency of the cell line being used to determine the titer. Therefore, several investigators use preferentially direct quantification for determining virus particle concentration. There are several bibliographic references for the counting of selected colonies (3,4). [ABSTRACT FROM AUTHOR]

The genera of the retroviridae comprise oncoretroviruses and lentiviruses (1). Oncoretroviruses can only infect dividing cells, as they require the breakdown of the nuclear membrane to access the cellular chromosomal deoxyribonucleic acid (DNA; ref. 2). Conversely, lentiviruses can also infect nondividing cells (3). Retroviral and lentiviral vectors are among the most powerful techniques for gene delivery into mammalian cells (1). For the sake of simplicity, we refer to retroviral and lentiviral vectors as retroviridae-based vectors. This chapter describes a method for the transient production of high titer, helper-free retroviridae-based vectors (4-6). The transient system for the production of retroviridae-based vectors is fast, reliable, and safe (1,4,5). In fact, the transient nature of this system greatly minimizes the possible formation of replication-competent viruses, which may occur via homologous recombination (1,4,5). The engineering of recombinant retroviridae vectors based on Moloney murine leukemia virus and HIV-1 is shown in Figs. 1 and 2, respectively (7). Retroviridae vector stocks can be easily generated in 48 h after the transfection of packaging cells. Three plasmids are simultaneously co-transfected into the packaging cells: one plasmid encodes for the viral envelope, a second plasmid encodes for the components of the viral core (gag-pol), and a third plasmid contains the transgene of interest, which could be either a reporter gene, a therapeutic factor, or a combination of both. The function of the third plasmid consists in providing the chimerical viral genome for the packaging into the recombinant retroviridae-based vector. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) radiation is mutagenic in a wide variety of organisms and is a major source of physical deoxyribonucleic acid (DNA) damage. The mutations caused and the biological consequences of exposing cell cultures to UV light have been extensively studied and have given many insights into the underlying mechanisms that lie behind DNA damage and repair (1-3). UV radiation occurs at three wavelengths: 400 to 320 nm (UV-A), 320 to 290 nm (UV-B), and 290 to 100 nm (UV-C). Although UV-C is generally accepted to be the most harmful source of UV radiation, it occurs mainly in artificial light, as opposed to UV-A and B, which occur naturally in the environment as solar radiation. [ABSTRACT FROM AUTHOR]

γ Radiation is an electromagnetic radiation with wavelengths in the range of 0.1-100 pm. Like all forms of electromagnetic radiations, the γ ray has no mass and no charge and interacts with material by colliding with the electrons in the shells of atoms. Irradiation with γ rays is very penetrating, and the energy transferred induces a wide variety of lesions into the components of cultured cells, especially deoxyribonucleic acid (DNA). [ABSTRACT FROM AUTHOR]

Alkylating agents are a series of potentially carcinogenic compounds that are able to introduce lesions into deoxyribonucleic acid (DNA; ref. 1). For their electrophilic nature, alkylating agents have an affinity for the nucleophilic centers of DNA and are monofunctional (have one reactive group) or bifunctional (have two reactive groups). Alkylating agents, such as methylmethanesulphonate and N-ethyl-N-nitrosourea, introduce methyl, ethyl, and complicated alkyl groups to the functional groups of nucleic acid bases, with ring nitrogens being generally more reactive than ring oxygens (2-4). The alkylation of oxygen in phosphodiester bonds results in phosphotriester formation (5) and bifunctional agents often react with two nucleophilic centers, producing inter- and intrastrand crosslinks (6). [ABSTRACT FROM AUTHOR]

Apoptosis plays a critical role in important biological processes such as morphogenesis, tissue homeostasis, and elimination of genomic damaged or virally infected cells and of self-reactive clones from the immune system (1). Although apoptosis is important during normal development, its aberrant activation may contribute to a number of diseases, such as AIDS, neurodegenerative disorders, and ischemic injury (2). However, impaired apoptosis may be a significant factor in the etiology of diseases like cancer, autoimmune disorders, and viral infections (2). [ABSTRACT FROM AUTHOR]

Apoptosis, or programmed cell death, refers to both the initiation and execution of the events whereby a cell commits suicide. This process is important in development and its deregulation is found in many diseases (1-6), including cancer (6-10). Apoptosis is distinct from other ways in which cells may lose viability (e.g., necrosis, senescence). Apoptosis is an active process triggered by a variety of stimuli, which induces closely comparable structural changes (Fig. 1). These morphological changes are especially evident in the nucleus where the chromatin condenses to compact and apparently simple, globular, crescent-shaped figures (11). Other typical features include cytoplasmic shrinkage, zeiosis, and the formation of apoptotic bodies within the nucleus. The earliest definitive changes in apoptosis that have been detected by electron microscopy are compaction of the nuclear chromatin into sharply circumscribed, uniformly-dense masses that abut on the nuclear envelope and condensation of the cytoplasm. Continuation of condensation is accompanied by convolution of the nuclear and cellular outlines, and nucleus often break up at this stage to produce discrete fragments. The surface protuberances then separate with sealing of the plasma membrane, converting the cell into a number of membrane-bounded apoptotic bodies of varying size in which the closely packed organelles appear intact; some of these bodies lack a nuclear component, whereas others contain one or more nuclear fragments in which compacted chromatin is distributed either in peripheral crescents or throughout cross-sectional area. [ABSTRACT FROM AUTHOR]

Malignant tumors are composed of cells that have lost their proliferation control and multiply independently of a physiologic need for increase in cell number in the tissue. Cells that have lost these proliferative controls are often referred to as "transformed." Transformation of a mammalian cell has been defined as the acquisition of permanent disturbances of growth and/or locomotion control (1). This definition of transformation implies a phenotypic change that has been found to be induced either by the introduction of a new gene into a cell or by the mutation of the genome through either irradiation or deoxyribonucleic acid (DNA)-damaging agents. Genes that can be introduced into normal cells to produce a transformed phenotype are usually derived from oncogenic viruses. Thus the definition of transformation in mammalian cells differs form the definition of transformation in bacteria where the term refers to the introduction of any new gene into the bacterial genome. [ABSTRACT FROM AUTHOR]

It is becoming apparent that an increase in the rate of protein degradation represents one mechanism for the regulation of key proteins during the cell cycle (1). The importance of protein degradation in the control of cell proliferation is elegantly illustrated by the regulated synthesis and destruction of the cyclin family of proteins during the life cycle of a cell. The cyclins are a group of proteins that influence the function of a series of intracellular kinases (the cyclin-dependent kinases or CDKs). The activation of specific CDKs is required during specific phases of the life cycle of a cell (this life cycle is known as the cell cycle). For example, the longest phase of the life cycle of a cell is the phase known as G1, or gap 1, which precedes the replication of cellular deoxyribonucleic acid (DNA) during S phase. Cells in G1 contain relatively high levels of two cyclins, known as cyclin D and cyclin E. These two cyclins are degraded as cells enter S phase and begin to replicate their DNA. The orderly synthesis and degradation of the cyclins is critical to proper progression of cells through the phases of the cell cycle. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle control and progression (1,2). A CDK holoenzyme complex is active if associated with its cyclin partner and if the complex is phosphorylated at specific activating residues (threonine 160/161; refs. 1 and 2). The progression through the cell cycle is mediated by the sequential activation of CDKs during different phases of the cycle. The G1/S phase transition of the cell cycle is mediated by phosphorylation of the Retinoblastoma gene product (Rb) by CDK4 and/or CDK6 (G1/ S checkpoint), leading to the release of bound E2F from Rb and allowing the transcription of genes by "free E2F" necessary for S-phase progression. During late G1 phase, the CDK2/cyclin E complex phosphorylates several substrates, including Rb. However, CDK2/cyclin A complex is active throughout the S phase. For cells to progress to mitosis, CDK1 (CDC2) must be "activated," and activated cdc2 phosphorylates substrates required for mitosis (3). [ABSTRACT FROM AUTHOR]

Ligation-mediated polymerase chain reaction (LM-PCR) is used for high-resolution genomic footprinting. This technique was originally developed to study in vivo protein-deoxyribonucleic acid (DNA) interactions at regions of genes important for transcriptional regulation. Successful analysis of in vivo occupancy of gene regulatory elements by LM-PCR largely depends on the strategy and quality of reagents. The experimental outline for LM-PCR reaction is shown in Fig. 1. For a through understanding of this procedure and other alternative options, see Ausubel et al. (1). [ABSTRACT FROM AUTHOR]

Mapping DNase I hypersensitive sites can often localize the control regions in a eukaryotic gene. It is generally believed that chromosomal regions with loose or more open conformation are sensitive to DNase I cleavage. By comparing the patterns of hypersensitive sites obtained by digestion of silent genes with those obtained when the genes are actively transcribed, regions that participate in gene regulation (either induction or suppression) can be identified. A major strength of this method is that it is an "unbiased" approach to characterize the location of control sequences for the regulation of a gene. A good example of the power of DNase I hypersensitivity mapping is the rat Osteocalcin gene. Osteocalcin gene expression is detected only in bone tissues and in fully mature osteoblasts. DNase I cleavage is not observed in the OC gene in nonbone tissues or in immature osteoblasts (where OC is not transcribed). However, two DNase I hypersensitive sites are detected in the OC gene in bone tissues and in mature osteoblasts where the gene is actively transcribed. The first DNase I region encompasses the basal promoter regions that contain regulatory sequences for the RUNX, C/EBP, activator protein-1, and TFIID transcription factors. The second DNase I hypersensitive domain is mapped to a region 0.5 kilobases upstream of the transcription initiation site. This region contains a vitamin D enhancer element and regulatory sequences for RUNX factors. The sequences at these hypersensitive sites are both sufficient and necessary for basal and bone-tissue specific expression of the Osteocalcin gene. [ABSTRACT FROM AUTHOR]

The electrophoretic mobility shift assay (EMSA) determines sequence-specific protein-deoxyribonucleic acid (DNA) interactions (e.g., transcription factors with cognate regulatory elements). This assay is based on reduced electrophoretic mobilities of protein/DNA complexes through a nondenaturing polyacrylamide gel compared with unbound DNA fragments or double-stranded oligonucleotides. If multiprotein complexes bind, the migration rate slows further with each additional protein. Oligonucleotide competition and antibody binding controls provide further evidence of binding specificity and protein identity. In competition experiments, unlabeled (cold) DNA or the same oligonucleotide (being used as a probe) with wild-type or mutated sequences of binding site or other unrelated binding sequences are added to the reaction. Interactions between the binding protein and the unlabeled related sequences would decrease the band intensity of the previously shifted complexes, whereas mutated or unrelated binding sequences will not change the intensity of the previously shifted complexes (an example is shown in Fig. 1). [ABSTRACT FROM AUTHOR]

Fundamental parameters of biological control require sequence-specific protein-deoxyribonucleic acid (DNA) interactions. The packaging of DNA into higher order chromatin controls accessibility to DNA binding proteins that influence regulatory events that include transcription, replication, recombination, and DNA repair. Chromatin is remodeled in a physiologically responsive manner by covalently modifying histones to neutralize a positive charge of these basic chromosomal proteins that form nucleosomes, the primary unit of nucleoprotein structure. Acetylation and methylation of residues on the unstructured tails of histone proteins are the most prevalent chromatin modification related to transcriptional regulation. To understand chromatin structure of a gene, it is important to characterize the interactions of structural and regulatory proteins in the vicinity of covalently modified nucleosomes. Formaldehyde crosslinking rapidly fixes protein-protein and protein-DNA complexes in vivo providing the basis for an approach to analyze native structure of gene promoters. The protocol below is a simple yet sensitive method to determine whether a known protein is associated with a DNA sequence in mammalian cells. It must be noted that this protocol is not limited to sequence-specific DNA binding proteins. The success of this procedure relies on the use of an antibody that specifically and tightly binds its target protein in the buffer, and wash conditions used for the chromatin immunoprecipitation (ChIP) assay. Briefly, the protein-protein and protein-DNA complexes in vivo are reversibly crosslinked by formaldehyde. [ABSTRACT FROM AUTHOR]

Only a handful of tagged molecules have been used successfully in vivo to monitor gene expression. The most commonly used are β-galactosidase (β-gal) and green fluorescent protein (GFP). Both molecules pose limitations with in vivo detection. When using GFP, autofluorescence may be encountered in tissues and tissue sections. Similarly, when using β-gal as a marker, the penetration of X-gal substrate is difficult in tissues with extensive extra cellular matrix and in calcified tissues. [ABSTRACT FROM AUTHOR]

The resolution that can be attained in assessment of the intranuclear localization of cellular proteins is dependent on specificity of the antibodies. Primary antibodies should be well characterized. We recommend testing antibody specificity by immunoblotting. In general, monoclonal antibodies have a selective advantage of recognizing only a single epitope but may not produce a high titer. In contrast, polyclonal antibodies generally have a high titer but may recognize multiple epitopes that may result in crossreactivity with several proteins because of similar amino acid sequences. The problem of low titer antibody and poor antigenicity can be overcome by marking the proteins with an epitope "tag." The most widely used tags in prokaryotic and eukaryotic cells include glutathione S-transferase , β-galactosidase, 6× histidine, hemagglutinin, Xpress, Flag, Myc, and a variety of fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein, yellow fluorescent protein, and red fluorescent protein). Caution must be exercised when tagging large proteins (e.g., GFP or β-galactosidase) at the amino or carboxy termini to avoid interference with folding that can influence activity. Most "epitope tags" are very short sequences, thereby having little or no crossreactivity with other cellular proteins. The well-characterized antibodies that are commercially available against these tags are also conjugated to fluorescent molecules. These conjugated reagents reduce both the time and assessment of chemical needed to visualize the final product. [ABSTRACT FROM AUTHOR]

Immunofluorescence is one of the most widely used techniques to study the localization of transcription factors, proteins, and structural components of nuclear architecture and cytoarchitecture. High-resolution in situ immunofluorescence approaches permit assessment of functional interrelationships between nuclear structure and gene expression that are linked to the intranuclear compartmentalization of nucleic acids and regulatory proteins (an example is shown in Fig. 1). The success of this method is dependent on the quality and specificity of the antibodies and the relative stability of antigens. Generally, the overall scheme for localization of cellular proteins involves fixation and permeabilization of cells for antibody accessibility, blocking, and staining with specific antibodies before microscopic examination. To reveal the subcellular and subnuclear macromolecular complexes that comprise and govern activation of the regulatory machinery for gene expression, cells can be subjected to selective extractions before immunodetection as described below. [ABSTRACT FROM AUTHOR]

In the 20 yr since the discovery of proteins whose levels oscillate during the cell cycle in marine invertebrate embryos (1), the study of cyclins and their cognate protein kinases has revealed a wealth of information on how eukaryotic cells control cyclical functions connected with cell proliferation and growth. The picture that has emerged from two decades of investigation is intricate and still incomplete. In the simplest possible model, cyclins are critical regulatory subunits of cyclin-dependent protein kinases (CDKs). When cyclin levels rise, they form stable complexes with CDKs, generating enzymatically active heterodimeric complexes. When cyclin levels fall, CDKs lose catalytic activity and are unable to phosphorylate their substrates. This simple model remains fundamentally valid, but it is now clear that the regulation of cyclin/CDKs is exquisitely complex throughout the cell cycle. Moreover, it is now widely recognized that cyclins and CDKs do much more than simply control cell cycle progression. The relatively simple mechanisms discovered in yeast cells, which have one G1 cyclin, one G2 cyclin, and a single CDK, are replaced in mammalian cells by a richly redundant molecular network, including multiple cyclins, CDKs, and regulatory pathways that cross-talk with a dizzying array of cell fate determination molecules. Thus, it is hardly surprising that initial hopes for quick discovery and therapeutic development of highly specific pharmacological inhibitors of cyclin/CDK complexes have not yet been fully realized. [ABSTRACT FROM AUTHOR]

The assessment of the viral titer is an important step in optimizing and reproducing working conditions for the gene transduction for various mammalian cell types. The assessment of the viral titer is an important parameter to determine the maximum number of target cells that can be infected for a given volume of viral stock (1). [ABSTRACT FROM AUTHOR]

This technique is used to visualize sites of active transcription in a permeabilized cell and does not require radiolabeled molecules (e.g., [3H] Uridine). Nonradioactive ribonucleic acid (RNA) precursors (e.g., 5-bromouridine-5′-triphosphate [BrUTP]) are used and can be detected by using fluorescently labeled antibodies. Procedures for BrUTP of labeling transcription sites require manipulations that are best applied to adherent cells but can be applied, with difficulty, to cell cultures in suspension. The protocol described below is for adherent cells grown on cover slips. [ABSTRACT FROM AUTHOR]