Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Lentiviruses are excellent molecular tools for expressing (or silencing) a gene of interest through infection of target cells, either in vitro or in vivo. Lentiviruses have relatively long incubation periods (lenti is Latin for slow), and are very efficient infectious agents. Lentiviral expression works by packaging the viral proteins together in plasmids with your gene of interest, a promoter, and perhaps selection or inducible expression markers. Lentiviruses infect by attaching to the cell membrane and releasing viral materials into the cell, where they easily traverse the nuclear membrane. Lentiviral expression systems are used in a range of applications, such as blocking gene expression with RNA interference, or in developing strategies for gene therapy.
Why use lentiviral expression?
Lentiviruses are excellent gene-delivery machines, for a number of reasons.
They can infect almost anything. Lentiviruses can infect (or transfect) both dividing and quiescent cells. This makes for much greater infection efficiency compared to other retroviruses, for example, which can only infect dividing cells. Lentiviruses are also happy to infect virtually any cell type, even difficult-to-transfect types such as primary cells. But rest assured, they have been rendered relatively safe to use (see below).
They are flexible and easy to work with. The production and manipulation of lentiviral vectors is relatively easy. Many complete kits are available from vendors that make this work faster, safer, and more convenient than ever. In addition, lentiviruses can be used for either stable or transient protein expression, with high efficiency.
Choosing the best lentiviral system
Vendors have capitalized on the utility of lentiviruses by offering a range of lentiviral expression systems. Given all the good reasons to use lentiviral expression, how do you go about choosing a system? Considering several points may help you choose.
Consider safety. Left to their own devices, lentiviruses can efficiently infect humans. To take advantage of their infection efficiency while also maintaining safety standards, scientists have developed a strategy in which the lentiviral elements are split into groups, with each group in a different vector. You should choose at least a 3rd-generation system, which are common today. Third-generation systems use three plasmids to separate key viral components: a packaging vector that contains structural and packaging genes, the VSV-G envelope vector, and the transfer vector that contains the gene of interest and sequences for RNA production. Viral genes that are not needed for packaging are also removed to prevent production of a replication-competent virus. These strategies greatly reduce the chance that the virus might reconstitute itself inside the cell and emerge replication-ready.
There are also lentiviral systems that divide the viral genes into 4 or 5 separate vectors, if necessary. Although the vectors have been rendered very safe, the National Institutes of Health (as well as lentiviral expression system vendors) recommend working with lentiviruses under Biosafety Level 2 conditions and Class II culture facilities as a precaution.
Consider expression boosters. A traditional drawback of lentiviral vectors is that they generally give low titers. But never fear – vendors of lentiviral expression kits have developed different ways to boost expression so that your viral titer numbers remain high. When choosing an expression system, though, you may want to ensure that such a mechanism is present. For example, including the VSV-G envelope protein in vectors helps to stabilize the viral particle, increase gene transfer efficiency for higher viral titers, and increase the number of cell types the virus can infect.
Consider promoters and selection markers. As in all expression systems, different types of expression promoters are available, and may work better in some cells over others depending upon your experimental conditions. Vendors also offer optional markers to indicate successfully infected cells. Some of the options available include fluorescent tags such as GFP, or the lacZ gene that produces beta-galactosidase.
Consider inducible elements. Having the power to induce gene expression, for example when you add a trigger substance to your cultures, may be a valuable addition to your experiments. Inducible lentiviral expression systems are available to turn expression off and on like a switch. One example uses a CymR repressor that binds a cumate operator sequence – the repression is removed by adding cumate, a small molecule that binds to CymR. Other systems use antibiotic-controlled transactivators that, for example, bind to a tetracycline response element located within the promoter for your gene of interest. Gene expression can be activated by the addition (or in some systems, the withdrawal) of antibiotic such as doxycycline.
Conclusion
The manifold options available for lentiviral expression make it easier to customize the expression tools for your experimental system. Add to that the extensive built-in safety features, and lentiviral expression systems are attractive and efficient viral tools for introducing genes into cultured cells or animal models.
The image at the top of the page is from System Biosciences.