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Using Nucleases to Stimulate Homologous Recombination

In essentially all organisms, double-strand breaks in chromosomal DNA stimulate repair by multiple mechanisms, including homologous recombination. It is possible to use site-specific reagents to produce a break or other recombinagenic damage at a unique site, which makes possible ...

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Gene Targeting at the Chromosomal Immunoglobulin Locus: A Model System for the Study of Mammalian Homologous Recombination Mechanisms

Plasmid DNA transfected into mammalian cells can integrate into mammalian chromosomes by homologous recombination. This phenomenon, known as gene targeting, can be used as a tool to investigate mammalian homologous recombination mechanisms. The chromosomal immunoglobulin ...

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Enhancement of In Vivo Targeted Nucleotide Exchange by Nonspecific Carrier DNA

Targeted nucleotide exchange (TNE) is a process in which an oligonucleotide bearing sequence complementarity aligns with the sequence of a target gene and directs the alteration of a single base. This technique can be used to repair a point mutation or mediate site-specific mutagenesis. A cr ...

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Chromatin Immunoprecipitation to Investigate Protein-DNA Interactions During Genetic Recombination

Chromatin immunoprecipitation is a technique that allows one to examine the in vivo localization of proteins to DNA. This technique is well suited for studying genetic recombination since it can provide both a temporal and spatial assessment of the dynamic association of proteins with DNA ...

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Holliday Junction Branch Migration and Resolution Assays

Holliday junctions are central intermediates in the process of genetic recombination; they form as a consequence of a reciprocal exchange of strands between paired DNA molecules. Enzymes that specifically recognize and process these junctions are necessary for the formation of rec ...

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The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA

In the search for methods to explore the interaction between proteins and DNA, a plethora of footprinting techniques have been developed, many of which are discussed elsewhere in the present work. Most footprinting techniques are based on the simple premise of specific DNA regions being pro ...

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CTL Analysis for Tumor Vaccines

Many studies have been conducted with the aim to stimulate a therapeutic immune response against tumors. In most cases, efforts have been directed toward the induction of tumor-specific cytotoxic T lymphocytes (CTL), because this T lymphocyte subpopulation is considered to play a major r ...

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Development of DNA Vaccines for Salmonid Fish

Vaccination of fish against many different pathogenic organisms has made it possible to rear Atlantic salmon in net pen cages and produce fish commercially around the world. In fact, vaccine use is critical for the continued growth of the aquaculture industry and researchers are continual ...

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Immunostimulatory DNA Sequences: An Overview

The biochemical and genetic properties of DNA have been thoroughly investigated, yet only recently has it been appreciated that DNA carries more information than simply a blueprint for the regulation and construction of proteins. Indeed, the immune systems of vertebrates appear to have ...

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The Use of Bone Marrow-Chimeric Mice in Determining the MHC Restriction of Epitope-Specific Cytotoxic T Lymphocytes

Plasmid DNA immunization has emerged as a promising vaccine strategy against infectious agents, as well as a potential intervention for the treatment of cancer, autoimmunity, and allergy (1). Until recently, however, the cellular events by which injected plasmid DNA elicits potent ant ...

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Intramuscular Injection of DNA Vaccines in Fish

The DNA-based immunization technology has only been applied to fish very recently. Though a preliminary study showing reporter gene expression in fish muscles was published in 1991 (1), the first demonstration of an immune response to plasmid-encoded antigen was not reported until five y ...

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DNA-Based Immunization of Neonatal Mice

Immunization of neonates carries three inherent questions: (i) Is the neonatal immune system mature enough to elicit an immune response, and if not, how early can immunization be carried out? (ii) If an immune response is not elicited, will tolerance be induced? (iii) Can offspring of immune mothe ...

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Veterinary DNA Vaccines

Vaccination has relied, in general, on two technologies for the production of antigenic material suitable for the generation of a protective immune response; live infectious agents and inactivated or subunit vaccine preparations. Live infectious agents generally are most effica ...

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Intramuscular and Intradermal Injection of DNA Vaccines in Mice and Primates

There are several different ways to administer plasmid DNA vaccines (1). Those most commonly used include intramuscular (i.m.) injection or intradermal (i.d.) injection of pure plasmid DNA (“naked” DNA), or biolistic introduction of DNA-coated gold particles into the epidermis with a “ge ...

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Methodology Used in DNA-Based Prophylactic and Therapeutic Immunization Against Hepatitis B Virus in Chimpanzees

Chimpanzees, because of their near-human nature, have special needs that must be met by those who carry out medical research with them. Perhaps the most important of these is the need for companionship. Chimpanzees kept alone become obviously depressed, and manifest stereotypic behavior s ...

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The Immunology of DNA Vaccines

The surprising observation that direct inoculation of an expression plasmid encoding a foreign protein into the skin of mice resulted in the induction of antibody responses, demonstrated that injection of “naked” DNA could result in antigen expression in an immunogenic form (1). This obs ...

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Repeated Use of Qiagen Columns in Large-Scale Preparation of Plasmid DNA

The preparation of large amounts of high-purity intact plasmid DNA is a significant expense in DNA vaccine research. In our laboratory, mice are typically each immunized by injection of 100 μg on four occasions, so that an experiment with 50 mice requires 20 mg DNA as a minimum. The Qiagen tip-10,000 (Gi ...

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Assuring the Quality, Safety, and Efficacy of DNA Vaccines

Scientists in academia whose research is aimed at the development of a novel vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a candidate vaccine becomes a licensed product. This chapter will provide an overview of the regulatory process and will ...

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DNA Vaccination: Tolerance and Autoimmunity

Infectious diseases result in significant morbidity and mortality worldwide. Preventing these infections is both a public health priority and the primary goal of vaccine research. The discovery that cell-mediated and/or humoral immune responses against viruses, parasites, ba ...

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Development and Characterization of Lyophilized DNA Vaccine Formulations

The potential applications of using plasmid DNA for immunization and other gene therapy approaches have been discussed in an increasing number of publications in the past few years. Injection of mouse muscle with naked DNA (plasmid DNA in saline) resulted in significant episomal express ...

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