Duplex DNA sequences in eukaryotic chromosomes, corresponding to the genome of a virus, that are transmitted from one cell generation to the next without causing lysis of the host. Proviruses are often associated with neoplastic cell transformation and are key features of retrovirus biology.
JoVE Immunology and Infection
1Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, 2Department of Pathology and Laboratory Medicine, Emory University
HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.
Published August 31, 2014. Keywords: Infectious Diseases, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
1Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ)
Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.
Published June 25, 2014. Keywords: Genetics, gene therapy, integrome, integration site analysis, LAM-PCR, retroviral vectors, lentiviral vectors, AAV, deep sequencing, clonal inventory, mutagenesis screen
1Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA), 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles (UCLA)
We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.
Published November 26, 2014. Keywords: Stem Cell Biology, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
1Divisions of Experimental Hematology and Cancer Pathology, Cancer Blood Disease Institute, Cincinnati Children's Hospital Medical Center
Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.…
Published December 7, 2014. Keywords: Genetics, JAK2, BCR/ABL, TKI, random mutagenesis, drug resistance, kinase inhibitors, in-vivo resistance,
1Department of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine
In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.
Published May 24, 2012. Keywords: Neuroscience, Cell culture, transduction, lentiviral vector, neuron, astrocyte, promoter, CNS, genetics
1Max Plank Institute for Molecular Cell Biology and Genetics, Dresden
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.…
Published May 29, 2008. Keywords: Cell Biology, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
1Department of Cell Biology and Virology, Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg
Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.
Published June 16, 2008. Keywords: Medicine, HIV, Cell Biology, Recombinase, provirus, HeLa Cells