An Improved Protocol for the Production of Lentiviral Vectors

Abstract

Summary Lentiviral vectors are an ideal gene-delivery system for large gene-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, due to their high packaging capacity and broad tropism. Here, we present a calcium phosphate-based protocol for lentiviral production and concentration for in vitro and in vivo use. This revised procedure has been optimized to ensure high viral titers and transduction efficiency and is scalable to meet specific production needs.

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells

“…Notably, cells that had been recently transduced grew substantially slower as compared with non-transduced populations at the same passage and density. This was expected to be related to viral load stress, and cells were supplemented with additional FBS to compensate [49]. Despite the increased FBS concentration, over culture time post-transduction cultures tended to see increases in the non-fluorescent cell sub-population percentage.…”

Extensible combinatorial CRISPR screening in mammalian cells

“…3 rd generation packaging plasmids can also be used. A detailed protocol for lentivirus production was described in ( Brown et al., 2020 ). The lentiviral-based plasmids that express wild-type SpCas9 (Addgene: #73857) and high-fidelity SpCas9 such as Opti-SpCas9 (Addgene: #131736) could be used for generating the stable SpCas9-expressing cell line.…”

Production of human CAR-NK cells with lentiviral vectors and functional assessment in vitro

“…However, the amount of total DNA added should be optimized using this method. Refer to Brown et al. (2020) and Follenzi and Naldini (2002) for protocols on LV production using calcium phosphate.…”