For cell types not amenable to lipid-mediated transfection, viral vectors are often employed. Virus-mediated transfection, also known as transduction, offers a means to reach hard-to-transfect cell types for protein overexpression or knockdown, and it is the most commonly used method in clinical research (Glover et al., 2005; Pfeifer and Verma, 2001). One of the main advantages of viral delivery is that the process can be performed inside a living organism (in vivo) or in cell culture (in vitro) with gene delivery efficiencies approaching 95–100%.
Viral vectors are tailored to their specific applications, but must generally share a few key properties:
Adenoviruses are DNA viruses with broad cell tropism that can transiently transduce nearly any mammalian cell type. The adenovirus enters target cells by binding to the Coxsackie/Adenovirus receptor (CAR) (Bergelson et al.,1997). After binding to the CAR, the adenovirus is internalized via integrin-mediated endocytosis followed by active transport to the nucleus, where its DNA is expressed episomally (Hirata and Russell, 2000). Although adenoviral vectors work well for transient delivery in many cell types, for some difficult cell lines such as non-dividing cells and for stable expression, lentiviral vectors are preferred. The packaging capacity of adenoviruses is 7–8 kb.
Retroviruses are positive-strand RNA viruses that stably integrate their genomes into host cell chromosomes. When pseudotyped with an envelope that has broad tropism, such as vesicular stomatitis virus glycoprotein (VSV-G), these viruses can enter virtually any mammalian cell type. However, most retroviruses depend upon the breakdown of nuclear membrane during cell division to infect cells and are thus limited by the requirement of replicating cells for transduction. Other disadvantages of retroviruses include the possibility of insertional mutagenesis and the potential for the activation of latent disease. Like adenoviruses, retroviruses can carry foreign genes of around 8 kb.
Lentiviruses are a subgroup of the retrovirus family; as such, they can integrate into the host cell genome to allow stable, long-term expression (Anson, 2004). In contrast to other retroviruses, lentiviruses are more versatile tools as they use an active nuclear import pathway to transduce non-dividing, terminally differentiated cell populations such as neuronal and hematopoietic cells.
Adeno-associated viruses are capable of transducing a broad range of dividing and non-dividing cells types, but they require coinfection with a helper virus like adenovirus or herpes virus to produce recombinant virions in packaging cells. This causes difficulties in obtaining high quality viral stocks that are free of helper viruses. Furthermore, adeno-associated viruses have only limited packaging capacity of up to 4.9 kb. On the other hand, adeno-associated viruses show low immunogenicity in most cell types, and they have the ability to integrate into a specific region of the human chromosome, thereby avoiding insertional mutagenesis.
Other viral vector systems that can be used for overexpression of proteins include vectors based on baculovirus, vaccinia virus, and herpes simplex virus. While baculoviruses normally infect insect cells, recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by including a dominant selectable marker in the baculoviral vector, cell lines can be derived that stably express recombinant genes (Condreay et al., 1999). Vectors based on vaccinia virus can be used for introducing large DNA fragments into a wide range of mammalian cells. However, cells infected with vaccinia virus die within one or two days, limiting this system to transient protein production. Herpes simplex viruses are a class of double-stranded DNA viruses that infect neurons.
|Viral System||Size||DNA insert size||Max titer (particles/mL)||Infection||Expression||Drawbacks|
|8 kb||1 × 1013||Dividing and non-dividing cells||Transient||Elicits strong antiviral immune response|
|8 kb||1 × 109||Dividing cells||Stable||Insertional mutagenesis potential|
|9 kb||1 × 109||Dividing and non-dividing cells||Stable||Insertional mutagenesis potential|
|Adeno-associated virus||8.5 kb|
|5 kb||1 × 1011||Dividing and non-dividing cells||Stable;|
|Requires helper virus for replication; difficult to produce pure viral stocks|
|2 × 108||Dividing and non-dividing cells||Transient or|
|Limited mammalian host range|
|Vaccinia virus||190 kb|
|25 kb||3 × 109||Dividing cells||Transient||Potential cytopathic effects|
|Herpes simplex virus||150 kb|
|30–40 kb||1 × 109||Dividing and non-dividing cells||Transient||No gene expression during latent infection|
For Research Use Only. Not for use in diagnostic procedures.