Nov 20, 2023
Lentiviruses have become indispensable tools in molecular biology, enabling precise gene delivery and manipulation. As researchers harness the potential of lentiviral vectors, it is paramount to prioritize biosafety. There have been considerable efforts to improve the safety of lentiviral vectors, thus 3 different generations exist, with increasing safety modifications. 2nd and 3rd generation systems are the most common vectors seen in the marketplace, thus knowing the difference is imperative.
This blog will explore the difference between 2nd and 3rd generation lentiviral systems, best practices for handling lentivirus, and optimizations made in OriGene’s lentiviral system to further enhance safety.
What is the difference between 2nd generation and 3rd generation Lentivirus?
2nd generation lentiviral systems utilize 3 plasmids for co-transfecting cells to produce lentiviral particles:
- A transfer plasmid containing the gene of interest (GOI)
- One plasmid containing the packaging genes Gag, Pol, Rev, and Tat
- One VSV-G envelope plasmid
3rd generation lentiviral systems utilize 4 plasmids for co-transfecting cells to product lentiviral particles:
- A transfer plasmid containing the GOI
- One plasmid containing the packaging genes Gag & Pol
- One plasmid containing the packaging genes Rev
- One VSV-G envelope plasmid
By splitting the packaging genes between 2 different plasmids and removing unnecessary genes such as Tat, we reduce the likelihood of developing replication competent lentivirus, making 3rd generation lentivirus safer than 2nd generation systems.
Best Practices for Handling Lentivirus
Although 3rd generation systems are optimized for safety, NIH guidelines recommend handling lentivirus under BSL-2 conditions due to their ability to transduce primary human cells. Researchers should don appropriate Personal Protective Equipment (PPE), including gloves, lab coats, and eye protection, to minimize the risk of skin contact or accidental exposure to aerosols during lentivirus handling. By strictly adhering to these best practices, researchers can mitigate potential risks associated with lentivirus handling and contribute to responsible and safe research practices.
It is crucial to note that while the provided best practices offer general guidelines for the safe handling of lentivirus, researchers must adhere to and prioritize the specific biosafety guidelines and protocols established by their respective institutions.
OriGene’s Lentiviral System
OriGene’s lentiviral vectors (shRNA and overexpression lentiviral vectors) are 3rd generation lentiviral vectors with improved biosafety. These safety features are discussed below:
- Our pLenti expression vector is replication deficient as it contains SIN (Self Inactivation), a deletion in the 3′ LTR (ΔU3). This SIN deletion does not affect lentiviral packaging, yet results in “self-inactivation” after integration into the transduced cell. The integrated lentiviral genome is no longer capable of self-replication.
- A constitutive promoter (CMV promoter) has been placed upstream of the 5′ LTR in the pLenti expression vector to offset the requirement for Tat in the efficient production of viral RNA.
- The packing plasmids (Gag, pol, rev and VSV-G) supporting lentiviral packaging are separated onto three plasmids; none of them contain LTRs or the Ψ packaging sequence. Only the lenti vector for gene overexpression or shRNA knockdown contains the packaging signal that can be packaged into viral particles.
- The VSV-G gene from Vesicular Stomatitis Virus is used as the pseudo-envelope, further reducing the chances of recombination with viral genome.
- The Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) is a regulatory element which is transcribed, but not translated. When WPRE is inserted in the 3' untranslated region (UTR) of a mammalian expression cassette, it can significantly increase mRNA stability and protein yield for genes delivered by viral vectors. Due to a lack of WPRE protein expression, the oncogenic potential is minimized/not present.
Lentiviral Solutions for Researchers
OriGene proudly offers a range of cutting-edge lentiviral products, providing researchers with the tools they need to unlock the full potential of this revolutionary technology.