Lentiviral particles can be an essential delivery tool for difficult-to-transfect cells. However, regulations regarding biosafety features can sometimes make the technology intimidating for a first-time user. What do the terms mean, and how can you determine if the components fit your institution’s safety requirements?
Components necessary for packaging
A lentiviral packaging reaction consists of a co-transfection of two main components: A transfer vector coding the product for your gene of interest (eg: SMARTvector shRNA for MYC, or Edit-R Lentiviral Blast-Cas9 Nuclease), and a packaging mix containing genes encoding other the elements necessary for packaging the lentiviral particles (eg: Dharmacon Trans-Lentiviral shRNA Packaging Kit). The largest potential risk often associated with lentiviral products is the concern of generation of recombinant viruses that are capable of autonomous replication; this is mitigated by alterations to the transfer vector and splitting the packaging components onto multiple plasmids, significantly decreasing the chance of viral self-replication and resulting in significantly increased biological security. Additionally, both of these components have their own safety features to consider.
Transfer vector features
For transfer vectors, the components of the plasmid itself are built for increased biosafety. First, all but the most essential elements of the lentiviral backbone have been eliminated from the vector (thus the requirement for a packaging mix to create lentiviral particles). Second, retained elements necessary for packaging and integration should be altered to mitigate unintended effects. For example, SMARTchoice lentiviral products contain a mutation of the 3’ long terminal repeat (LTR), rendering the vector self-inactivating, and reducing the probability of producing recombinant particles.
Packaging mix considerations
Due to the fact that the packaging mix will provide the gene components required to package the contents of the transfer vector into a lentiviral particle, additional safety features are built into this as well. When choosing your lentiviral packaging components, for optimal safety, it is advised to use the most advanced generation packaging mix that is compatible with your transfer vector. The standard definitions are:
- 2nd generation systems contain an all-in-one packaging vector expressing the Tat gene.
- 3rd generation systems do not express Tat, and the packaging is split in two or more plasmids.
- 4th generation systems express Tat, but are split across many more plasmids to further decrease the risk of recombination inherent to original 2nd generation mixes.
Dharmacon’s Translentiviral Packaging Kit is an example of a 4th generation packaging mix in which gene functions that facilitate the packaging process (such as those encoded by the structural genes gag, pol, env, etc.) are distributed between 5 separate plasmids which do not contain significant regions of homology. This strategy further minimizes the probability of recombination events that might generate viruses capable of autonomous replication (Wu et al 2000).
Conclusions
Whether you are interested in using a lentiviral particle format to knock down a gene or overexpress one, Dharmacon has options that can help you meet the most advanced safety requirements. While institutional regulations may vary, with these safety measures in place, lentiviral particles made using Dharmacon’s lentiviral packaging products can be used in standard Biosafety Level 2 tissue culture facilities. For further information about the safe handling of lentiviral particles, and biosafety considerations, see:
- U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL), Fifth Edition, Feb 2007.
- NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines), September 2009.
Reference
- Wu X., Wakefield J.K., Liu H., Xiao H., Kralovics R., Prchal J.T., Kappes J.C. Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther. 2000; 2(1):47-55.