A protocol for the production of integrase-deficient lentiviral vectors for CRISPR/Cas9-mediated gene knockout in dividing cells
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© 2017 Journal of Visualized Experiments. All rights reserved. Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.
Published Version (Please cite this version)10.3791/56915
Publication InfoVijayraghavan, S; & Kantor, Boris (2017). A protocol for the production of integrase-deficient lentiviral vectors for CRISPR/Cas9-mediated gene knockout in dividing cells. Journal of Visualized Experiments, 2017(130). 10.3791/56915. Retrieved from http://hdl.handle.net/10161/15930.
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Associate Research Professor of Neurobiology
Kantor Lab, the Duke University Viral Vector Core, facilitates the use of virus-mediated tools for gene transfer by investigators across diverse fields of study such as systems neuroscience, stem cell biology, metabolism, aging, cancer biology and others. Services are open to researchers within the Duke University Medical Center, Duke University, as well as outside investigators, worldwide.