Proviral Dna Integration
Proviral Dna Integration - Related Videos
10:20
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
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Cited by 2 •
2008
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.
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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Cited by 56 •
2016
We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.
07:58
Cell-Free DNA Integrity Analysis in Urine Samples
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Cited by 16 •
2017
A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.
14:27
CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
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Cited by 6 •
2018
We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.
10:58
Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma
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Cited by 44 •
2015
Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.
FAQs
Integrase provides the enzymatic connection between viral DNA and the host chromosome. It first processes the ends of the double-stranded DNA produced from viral RNA, then joins those ends to host chromosomal DNA. Cellular repair pathways complete the insertion, so integrase initiates a process that the host cell helps finish.
Conversion of viral RNA into double-stranded DNA creates the DNA substrate required for chromosomal insertion. The viral genome therefore passes through a change in molecular form before integrase acts. This sequence links reverse transcription to establishment of a provirus and explains why integration is studied as part of retroviral replication.
The chromosomal position of an inserted provirus matters because integration can have effects on host gene regulation. Examining those sites lets researchers move beyond asking whether insertion occurred and investigate where it occurred in relation to the host genome. That information is relevant to viral biology and the evaluation of integrating vectors.
A useful workflow follows viral RNA through reverse transcription, production of double-stranded DNA, integrase-mediated end processing, joining to host chromosomal DNA, and completion by cellular repair pathways. Tracking these stages separates viral and cellular contributions to insertion and helps connect the initial replication event with the resulting persistent provirus.
Integration-site analysis provides information about the chromosomal locations occupied by proviral DNA. Researchers can use those locations to investigate retroviral replication and insertional effects on gene regulation, while also applying the same analysis to questions in HIV biology. Site information therefore connects molecular events with consequences in the host genome.
Integrating gene therapy vectors require attention to where their DNA becomes inserted, not only whether delivery occurs. Integration-site studies support vector design and safety assessment by revealing the chromosomal context of insertion. This matters because the source material identifies insertional effects on gene regulation as a central concern when integration is used therapeutically.