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`Home › Technical Reference Library › Gibco Cell Culture Basics › Transfection Techniques & Basics › Gene Delivery Technologies › Viral Transfection › Viral Vectors
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`Viral Vectors
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`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%.
`
`Learn more about Selecting a Viral DNA Delivery System
`Key properties of viral vectors
`
`Viral vectors are tailored to their specific applications, but must generally share a few key properties:
`
`Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a
`part of the viral genome critical for viral replication, allowing the virus to efficiently infect cells and deliver the viral payload, but preventing the production of new virions in the absence of a helper
`virus that provides the missing critical proteins. However, an ongoing safety concern with the use of viral vectors is insertional mutagenesis, in which the ectopic chromosomal integration of
`viral DNA either disrupts the expression of a tumor-suppressor gene or activates an oncogene, leading to the malignant transformation of cells (Glover et al., 2005).
`Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects. This is especially important in studies requiring gene delivery in vivo, because the organism will
`develop an immune response if the vector is seen as a foreign invader (Nayak and Herzog, 2009).
`Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector.
`Therefore, unstable vectors are usually avoided.
`Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to
`target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudotyped.
`Selection: Viral vectors should contain selectable markers, such as resistance to a certain antibiotic, so that the cells that have taken up the viral vector can be isolated
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`Common viral vectors
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`Adenoviruses
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`Retroviruses
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`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
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`Adeno-associated viruses
`
`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.
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`TRANSGENE/BIOINVENT
`EXHIBIT 1031
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`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
`
`Adenovirus
`
`Retrovirus
`
`Lentivirus
`
`Adeno-associated
`virus
`
`Baculovirus
`
`Vaccinia virus
`
`Herpes simplex virus
`
`36 kb
`(dsDNA)
`
`7–11 kb
`(ssRNA)
`
`8 kb
`(ssRNA)
`
`8.5 kb
`(ssDNA)
`
`80–180
`kb
`(dsDNA)
`
`190 kb
`(dsDNA)
`
`150 kb
`(dsDNA)
`
`8 kb
`
`8 kb
`
`9 kb
`
`5 kb
`
`1 × 10
`13
`
`Dividing and non-dividing
`cells
`
`Transient
`
`Elicits strong antiviral immune response
`
`1 × 10
`9
`
`Dividing cells
`
`Stable
`
`Insertional mutagenesis potential
`
`1 × 10
`9
`
`1 × 10
`11
`
`Dividing and non-dividing
`cells
`
`Dividing and non-dividing
`cells
`
`Stable
`
`Insertional mutagenesis potential
`
`Stable;
`site-specific
`integration
`
`Requires helper virus for replication; difficult to produce pure viral
`stocks
`
`no known
`upper limit
`
`2 × 10
`8
`
`Dividing and non-dividing
`cells
`
`Transient or
`stable
`
`Limited mammalian host range
`
`25 kb
`
`3 × 10
`9
`
`Dividing cells
`
`Transient
`
`Potential cytopathic effects
`
`30–40 kb
`
`1 × 10
`9
`
`Dividing and non-dividing
`cells
`
`Transient
`
`No gene expression during latent infection
`
`For Research Use Only. Not for use in diagnostic procedures.
`
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