Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives

Abstract

The use of third-generation lentiviral vectors is now commonplace in most areas of basic biology. These systems provide a fast, efficient means for modulating gene expression, but experimental design needs to be carefully considered to minimize potential artefacts arising from off-target effects and other confounding factors. This review offers a starting point for those new to lentiviral-based vector systems, addressing the main issues involved with the use of lentiviral systems in vitro and outlines considerations which should be taken into account during experimental design. Factors such as selecting an appropriate system and controls, and practical titration of viral transduction are important considerations for experimental design. We also briefly describe some of the more recent advances in genome editing technology. TALENs and CRISPRs offer an alternative to lentivirus, providing endogenous gene editing with reduced off-target effects often at the expense of efficiency.