Go Viral with Your Research

To deliver a stretch of exogenous nucleic acid into a cell, chemical transfection and electroporation have been the most commonly used and adopted methodologies. As the challenges of cellular regulation continue to be deciphered, neuronal, stem, primary and some non-dividing eukaryotic cells are becoming the go-to experimental systems. Unfortunately, these cell types tend to be recalcitrant when used with such standard transfection methods. Researchers either abandon their studies or accept low transfection efficiencies and questionable results. There is another option for scientists to carry out their challenging recombinant studies, and that is viral transduction.

Viral vectors harness what nature has evolved over eons: the ability to insinuate foreign genetic material into a cellular fortress. The modern laboratory has tamed the virus, to help assure that it is both safe and efficacious, while exploiting its ability to transduce a very high percentage of cells (compared with transfection), says John Elder, professor of immunology and microbial science and director of protein expression at the Protein Expression and Proteomics Core facility at The Scripps Research Institute.

In progressing from the Wild West days into the twenty-first century, the field has largely coalesced around three distinct vehicles based on adenovirus (Ad), adeno-associated virus (AAV) and lentivirus (lenti), each with its own distinct virtues and vices. To assist researchers, we look at different aspects of viral vectors, what features distinguish each type and what applications they are best suited for.

Making virus

Using viruses as a vector, let alone making them, may sound scary. Yet scientists have come up with a variety of precautions—such as physically keeping the genes and key regulatory elements needed for propagation separate from the payload, so they are not packaged into viral particles that uncontrollably infect and replicate. Also, using proper biosafety equipment and best standard practices for working with viruses mitigates the chance of viral vectors doing any harm. These replication-deficient vectors have become standard tools for laboratory tasks as varied as intracellular imaging, gene editing and protein production.

Researchers can obtain ready-to-use, off-the-shelf or custom vectors from a variety of suppliers. They are typically delivered as a viral stock—a finite quantity of ready-to-use viral particles that can be put directly onto cells or injected into an animal, says X.D. Shao, chief operating officer of Vector Biolabs. Because Ad (unlike lenti and AAV) can be replicated in special packaging cells engineered to complement the missing Ad genes, it is sometimes delivered as “viral seed stock”—which the scientist can use to propagate and generate a continuing supply of vector material.

Another way to obtain viral vectors is to purchase plasmids that contain the essential regulatory elements and a promoter (inducible in some cases) as well as optional tags to assist in visualization or purification as the user generates the viral particles. Many of these plasmid constructs are ready to use, containing a specific gene of interest (GOI), or they are designed for RNAi, shRNA or CRISPR/Cas9 applications. These plasmids can be found as off-the-shelf products or custom ordered from many different suppliers, such as Vector Biolabs, GeneCopoeia and Cell Biolabs.

Making viral vectors requires some understanding of molecular biology, cell biology and virology, “and if the lab has all of this expertise in-house, they can certainly make the virus themselves,” says Shao. Kits that provide everything necessary to go from GOI to vector are available from several vendors.

Step by step

A typical kit-based workflow scenario might be: A GOI encoding the protein you want to express, for example, or RNA for a gene you’re looking to knock down, is cloned into a plasmid coding for some sort of selection, such as antibiotic resistance. The plasmid is transformed into bacteria, plated and a positive clone is selected and confirmed to contain the GOI. Next the clone is expanded, and the plasmid is harvested. This plasmid is then, in turn, used to co-transfect a producer cell—often a derivative of human embryonic kidney (HEK) cells—along with some other plasmid to make the viral particles that are ultimately used to transduce your cells of interest. Depending on the system you’re working with, there may be an additional cloning step or two involved.

Lentivirus will be secreted into the supernatant, and “you can just collect that ‘sup’ and use it straight [as is],” says Melissa Kelley, senior scientist in the R&D department of Dharmacon, part of GE Healthcare. The supernatant will generally contain cellular debris and may not be of a very high titer, so many researchers will purify and concentrate it. “It just depends on your application and the sensitivity of the cells you’re going to transduce in the end.” For AAV and Ad, virus is harvested by subjecting the producer cells to multiple rounds of freezing and thawing, followed by low-speed centrifugation.

The standard method to concentrate and purify virus is ultracentrifugation—which is laborious, low throughput, time consuming and technically challenging. “It’s not an industrial process,” says Pascale Bouillé, CEO of Vectalys, which offers ready-to-use lentiviral particles both directly and through the Takara Clontech catalog. Vectalys uses ultrafiltration to produce vector with titers of 109 infectious particles per milliliter. For vectors destined for “in vivo applications, we use a supplementary purification step based on chromatography,” Bouillé adds. She cautions that not all vendors deliver the same purity or titer of vector, and that customers should pay attention to the way titer is measured (e.g., transducing units, plaque-forming units or infectious particles), as well.

Labs producing their own viral vectors can take advantage of purification kits from several vendors, including Takara Clontech, Cell Biolabs, Applied Biological Materials and Sartorius. Bouillé sees these as best suited to academic users needing only a small quantity of vector.

Ad vs. AAV vs. lenti

There are many similarities among the three vector types, including the fact that all are capable of transducing both quiescent and dividing cells, and they can be pseudotyped to infect most cell types. There are several major distinctions among them, as well. With lenti, the virus integrates into the cell’s genome, but Ad and AAV typically remain episomal. Between Ad and AAV, the former is used primarily in vitro, but AAV is preferred in vivo. Generally speaking, AAV can only incorporate small inserts—on the order of 4.5 kb—but Ad can package about 8 kb, and lenti can handle about 10 kb.

The protocols for lenti and Ad are fairly well defined, and there’s not an advantage of using one over the other from a benchwork perspective, says Kelley. “It really comes down to what are the requirements of the experiment—which format would be most amenable based on the desired outcome.”

For example, lenti is capable of producing a stable cell line without the need for selection; however, there is a small risk that the integration will cause a deleterious mutation. On the other hand, Ad will express other genes. That’s the principal reason it’s out of favor for in vivo use—and even in vitro, as the vector can induce a lot of cellular responses independent of the GOI being transfected, points out Bouillé.

Neuronal, stem and primary cell lines have become essential tools for scientists seeking a better understanding of the in vivo cellular regulatory mechanisms of gene expression. However, these cell types have traditionally been difficult to transfect by classical methods. So, if you are trying to examine expression of your GOI but can’t seem to get the DNA into your cells, don’t be scared and settle for mediocre results. Let your research go viral!

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