Protocols in Current Issue
    Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome
    Authors:  Kinjal Majumder, Maria Boftsi and David J Pintel, date: 03/20/2019, view: 667, Q&A: 0
    [Abstract] The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by ...
    Nab Escaping AAV Mutants Isolated from Mouse Muscles
    Authors:  Zheng Chai, R. Jude Samulski and Chengwen Li, date: 05/05/2018, view: 2167, Q&A: 0
    [Abstract] Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These ...
    Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting
    [Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol ...
    Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
    Authors:  Giel Vandemoortele, Delphine De Sutter and Sven Eyckerman, date: 04/05/2017, view: 11019, Q&A: 0
    [Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair ...
    Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy
    [Abstract] Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the ...

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