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Identification of T Cell Epitopes of Mycobacterium tuberculosis with Biolistic DNA Vaccination

Tuberculosis (TB) has been listed as one of the most prevalent and serious infectious diseases worldwide. The etiological pathogen of TB is Mycobacterium tuberculosis (Mtb), a facultative intracellular bacterium. Mycobacterium bovis bacillus Calmette-Gu�rin (BCG) is the only ap ...

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Biolistic DNA Vaccination Against Trypanosoma Infection

Immunization to protect against Trypanosoma cruzi infection has the potential to greatly decrease the burden of Chagas’ disease in the Americas. Several target antigens have been explored by multiple investigators and show promise, but given that this parasite has multiple stages wi ...

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Biolistic DNA Vaccination Against Melanoma

We describe here the use of particle-mediated gene transfer for the induction of immune responses against melanoma antigens in murine tumor models using the melanocyte differentiation antigen tyrosinase-related protein 2 (TRP2) as an antigen in a murine B16 melanoma model. We have util ...

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Safety Assessment of Biolistic DNA Vaccination

DNA-based vector systems have been widely studied as new modalities for the prevention and treatment of human diseases. As for all other medicinal products, safety is an important aspect in the evaluation of such products. In this chapter we reflect on the basic safety issues which have been raised ...

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Efficiency of Biolistic DNA Vaccination in Experimental Type I Allergy

Gene gun-mediated delivery of allergen-encoding plasmid DNA has been in focus for many years now as being a needle-free alternative to the protein-based desensitization regimen used in specific immunotherapy. Biolistic immunization with the Helios gene gun has proven to be potent in the ...

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DiOlistics: Delivery of Fluorescent Dyes into Cells

DiOlistic labeling utilizes a particle-mediated delivery system to incorporate dye into cells. Because of its random nature, this technique generates sparse fluorescent labeling which is well suited for the study of neuronal dendritic branching and dendritic spine morphology. D ...

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Histone H2AX Phosphorylation: A Marker for DNA Damage

The DNA damage response can be initiated in response to a variety of stress signals that are encountered during physiological processes or in response to exogenous cues, such as ionizing radiation or DNA-damaging therapeutic agents. A number of methods have been developed to examine the mor ...

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Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40 Based In Vitro Assay of DNA Replication

The deleterious effects of DNA damage on DNA replication have been demonstrated in many model systems and the mechanisms of the resulting inhibition have been a research focus for at least 40 years. Moreover, recent studies have identified several major components of the S-phase checkpoint, ...

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In Vitro PCNA Modification Assays

Modification of the replicative sliding clamp, PCNA, by monoubiquitin, polyubiquitin, and SUMO contributes to the processing of DNA damage during replication. In order to investigate the properties of the relevant conjugation enzymes, their interactions, substrate recognit ...

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Detection of PCNA Modifications in Saccharomyces cerevisiae

PCNA modifications by members of the ubiquitin family are associated with a range of different transactions during replication of damaged and undamaged DNA. This chapter describes detailed protocols for the detection and isolation of ubiquitin and SUMO conjugates of PCNA from total b ...

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Quantitative Measurement of Translesion DNA Synthesis in Mammalian Cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism, in which specialized low-fidelity DNA polymerases bypass lesions that interfere with replication. This process is inherently mutagenic due to the miscoding nature of DNA lesions, but it prevents double strand brea ...

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Assays of Bypass Replication of Genotoxic Lesions in Cell-Free Extracts

The in vitro replication assay described here measures bidirectional replication of a circular double- stranded DNA template upon initiation at the SV40 origin. It models a single eukaryotic replication unit (replicon) and recapitulates the biochemical steps involved in the cata ...

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An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides

DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human ...

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In Vitro Rejoining of Double Strand Breaks in Genomic DNA

Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end joining (NHEJ) pathways in higher eukaryotes, and have facilitated the functional characterization of several of its components including DNA-PKcs, Ku, DNA ligase IV, ...

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Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae

Fluorescence microscopy of the DNA damage response in living cells stands out from many other DNA repair assays by its ability to monitor the response to individual DNA lesions in single cells. This is particularly true in yeast, where the frequency of spontaneous DNA lesions is relatively low co ...

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Measurement of Diversification in the Immunoglobulin Light Chain Gene of DT40 Cells

The immunoglobulin loci of the genetically tractable chicken B cell line DT40 provide a unique opportunity to study the cellular response to endogenously generated DNA damage in a chromosomal context. Abasic sites generated by the concerted action of Activation-Induced Deaminase ( ...

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Electrophoretic Mobility Shift Assays for ProteinDNA Complexes Involved in DNA Repair

The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the antic ...

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Establishment of the DNA Repair-Defective Mutants in DT40 Cells

The chicken B cell line DT40 has been widely used as a model system for reverse genetics studies in higher eukaryotes, because of its advantages including efficient gene targeting and ease of chromosome manipulation. Although the genetic approach using the RNA interference technique has b ...

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Isolating Genes Involved with Genotoxic Drug Response in the Nematode Caenorhabditis elegans Using Genome-Wide RNAi Screening

The soil nematode Caenorhabditis elegans has become a popular genetic model organism used to study a broad range of complex biological processes, including development, aging, apoptosis, and DNA damage responses. Many genetic tools and tricks have been developed in C. elegans including ...

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Identification of Genes Required for Damage Survival Using a Cell-Based RNAi Screen Against the Drosophila Genome

Exposure to DNA-damaging agents invokes biological responses necessary for damage recovery and cell survival. Despite the presence of intact DNA repair pathways, lack of certain other biological pathways has been shown to sensitize cells to DNA-damaging agents’ exposure. It is likely ...

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