Lentivirus is the most common gene delivery vehicle that is used to establish stable cell lines. After the lentivirus infects the host cell, its RNA genome is first reverse transcribed to DNA and integrated into the host’s genome in a seemingly random fashion. The chosen integration site has significant consequences for both the expression of your transgene and the phenotype of the host cell. Its integration mechanism could affect cellular function if the integration happens within a key gene or near oncogenes.
Figure 1. The lentivirus integration reaction.
Furthermore, lentiviruses are one of the backbones of gene therapy approaches to correct defective genes and treat diseases. Analysis of integration sites during gene therapy enables monitoring of possible adverse events and guides the development of safer integrating vectors. The adverse events reported from the X-linked severe combined immunodeficiency (X-SCID) gene therapy and other trials have resulted in reassessment of the risks associated with viral vector insertional mutagenesis. Thus, the elucidation of vector insertion sites is important for the future of gene therapy applications.
QVirus™ Platform has made a great effort for detecting and sequencing unknown DNA flanking sequences at vector integration sites. These efforts are generally based on PCR amplification of target sites. In addition, large-scale analysis of vector integration sites has been initiated with the next generation sequencing technologies. Our analysis service can help you identify the exact site of integration of recombinant lentiviral vectors.
Linear-amplification mediated PCR (LAM-PCR) allows identifying and characterizing unknown flanking DNA adjacent to known DNA of any origin. LAM-PCR has been developed to localize viral vector integration site (IS) within the host genome. As with transposons or retroviruses, genetic elements integrate their genome into the host genome in a (semi-) random manner. In many instances, it is decisive to know exactly the position where these vectors integrated.
Targeted sequence capture of unique regions is a novel approach that can be easily adapted to many genomic applications. Sequence capture enrichment is an amplification-free method which uses labelled, complementary DNA probes to hybridize the region of interest. Then the captured DNA is sequenced. Targeted sequence capture technology can be applied to the capture of lentiviral vector integration sites.
QVirus™ Platform is now offering a Next Generation Sequencing approach to specifically identify lentiviral integration sites in your lentiviral infected cells. Our method has been shown to be sensitive and specific for lentiviral sequences. The lentivirus integration site sequencing services include:
QVirus™ Platform's technical support team will be happy to work on confirming the details of your lentivirus integration site analysis service. If you have any special requirements, please feel free to contact us. We are looking forward to working together with your attractive projects.
1. Ustek D, et al. A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology. Infection Genetics and Evolution, 2012, 12(7):1349---1354.
2. Ronen, Keshet. Lentiviral Integration Site Targeting: Host Determinants and Consequences. Dissertations & Theses - Gradworks, 2010.