Gene therapy/mutation has become a widespread phenomenon today with the prospects of healthcare improvements and better disease control. Experts are attempting to discover better methods to change an organism’s genes to combat disabilities and cure inherited diseases and disorders. And lentiviral vectors can allow gene therapists to easily insert, delete, and modify an organism’s genes. This guide explains some crucial questions about lentiviruses, their usages, and their effectiveness in genetic engineering for beginners.
What are lentiviruses?
You may have heard of HIV, which causes AIDS and affects 1.2 million Americans. It belongs to a family of viruses called lentiviruses, which are classified as a genus of retroviruses. Retroviruses, as the name indicates, change the host’s DNA with a DNA replica of their RNA genomes. Some retroviruses can cause cancer among human beings, while other retroviruses, i.e., lentiviruses, lead to immunodeficiency. A lentivirus can be identified by its lengthy incubation period in different mammalian creatures, including:
- Apes
- Cats
- Cows
- Horses
- Humans
What makes lentiviruses useful?
Even though they are responsible for diseases such as AIDS in the past, humanity can find ways to incorporate lentiviruses into making gene vaccines. Since they can easily integrate heavy doses of cDNA into a host’s DNA, experts prefer using them for gene delivery. Also, one can observe how lentiviruses can become a part of the host’s germline genome, thereby passing down to the host’s descendants. These attributes make lentivirus-based vaccines more effective and powerful.
How to get the right packaging?
Lentiviral production becomes convenient when you’re using third-generation packaging for this reason. You should invest in Research Grade Lentiviral Vector Packaging to achieve the permanent integration of a gene into the host. So, getting a third-gen vector platform can help you produce the desired products in four weeks. Hence, with their multiple-plasmic approach and excellent transduction performance, these research-grade packaging materials offer a success rate as high as 96%! In other words, this often-tested packaging platform gives you functional viruses in laboratory environments. Normally, experts suggest a biotechnician use 293T cell lines to replicate lentiviruses better and keep them intact after creation.
Can lentiviruses make stable cell lines?
You can use lentiviruses to create stable lines, just like you would do with other retroviruses. Lentiviral genomes have successful selection markets, such as puromycin resistance genes containing the required antibiotics to kill off all cells that have resisted the adoption of the virus.
Lentiviruses are favored because of their higher stability and a consistent frequency of insertional mutagenesis. Hence, these vectors can easily work with even dendritic cells, thereby being ideal in immunotherapy vaccines.
What do we mean by MOI?
It’s described as the “Multiplicity of Infection” or the ratio between infection particles and infection targets. So, if you introduce 1,000 lentiviral particles to 100,000 healthy cells, 0.01 here is the MOI. This MOI can be different based on what cell lines you use. For instance, Jurkat has a higher MOI need (10) than MDA-MB-231 does (1). Finding the optimal MOI can help biotechnicians integrate viruses better.
What’s the life cycle of lentiviruses?
These particles either fuse with the cell’s membrane and enter the host or leverage endocytosis (when cells engulf external materials) to invade. After entering it, the particle begins to transcribe its cDNA onto the host’s nucleus in a process called ‘integration’. Some structural proteins will propagate the virus’ genetic material into the cell’s surroundings. In other words, you can explain the life cycle of this virus in six tiny steps that have been described like this by experts:
- Entering its host
- The viral genome spreads
- Reverse transcription happens
- The pro-viral DNA is then integrated
- Viral proteins get synthesized and assembled
- Exported viral genomes are exported out of the host
What is the best temperature for storing viruses?
They should be stored at – 80°C immediately after arrival to sustain their viability. After two/three cycles of freeze-thaw, we don’t recommend using these lentiviral particles in any of your average studies.
In laboratory terms, vector titer means the amount (or number) of viruses needed to infect the host you have present in a certain volume. Experts have described two titration methods such as functional and physical. In the first one, you determine transducing units and the number of colony-forming units after antibiotic selection. In the second one, you determine the number of p24 antigens and the genomic RNA concentration in the mixture.
Many research papers in the 21st century have shown that lentiviral particles can be attached to trastuzumab – an antibody used to treat different forms of cancer – and inserted into the patient’s body to weaken and destroy cancer.
How do we differ between 2nd and 3rd generations?
There are some crucial differences between second-generation and third-generation lentiviral systems. For instance, second-generation systems are marked by wild-type LTV viral promoters packaged in only one plasmid. On the other hand, third-generation systems feature hybrid promoters and contain not just one but two packaging plasmids. Moreover, third-generation systems can be packaged by two-gen too. It’s also safer since it uses three separate plasmids and doesn’t require that as the two-gen one does.
What are some safety issues associated with lentiviruses
Biosafety becomes a concern among genetic engineers inside a laboratory where these viral vectors are being produced. We recommend using three-gen systems to ensure fewer chances of a particle breaking out of its enclosure. The multiple-plasmid method ascertains that the genes needed to create packaged lentiviruses are never kept inside a single plasmid. These particles become replication-incompetent and cannot replicate themselves to cause a viral spread. But, again, you must be careful about these little particles contaminating the laboratory environment. So, regular testing can prevent any large-scale and dangerous problem from happening. So, purchase lentiviral packaging from trusted online vendors.
Conclusion
This article answers some queries regarding using lentiviral vectors in genetic engineering. We have discussed how lentiviruses can be integrated to make gene vaccines as these viruses can move on to the host’s germline, thereby making not only the host but the host’s descendants immune from a certain disease as well. You just have to buy reliable, quality packaging material to produce the desired lentiviral products. Invest in research-grade third-gen packaging to ensure you get the required results in a few weeks with functional lentiviral particles.
