Adenoviral Cloning Vectors

Kishan Sharma M.Sc. 1st Year Biotechnology Manipal University Jaipur

Cloning vector

A cloning vector is a small piece of DNA taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.

The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector.

For example by treating the vector and the foreign DNA with a restriction enzyme that cuts the DNA. DNA fragments thus generated contain either blunt ends or sticky ends.

Viral Vectors

Viral vectors are tools commonly used to deliver genetic material into cells.

This process can be performed inside a living organism or in cell culture.

Delivery of genes by a virus is termed transduction and the infected cells are described as transduced.

Properties of a viral vector

Safety: Viral vectors are 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.

Such a virus can efficiently infect cells but once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions.

Properties of a viral vector

Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects.

Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. The viral receptor can be modified to target the virus to a specific kind of cell.

Identification: Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Markers. A common marker is antibiotic resistance to a certain antibiotic.

Adenoviruses

Adenoviruses can be converted into efficient gene transfer vehicles.

Adenoviral vectors are not inherently dangerous. The dose of vector delivered is related to the toxicity observed.

Adenoviruses

Icosahedral shape.

Composed of 13% DNA & 87% Protein.

Nonenveloped viruses with an icosahedral nucleocapsid containing a double stranded DNA genome.

Size 90-100 nm.

Adenoviruses

Since humans commonly come in contact with adenoviruses which cause respiratory, gastrointestinal and eye infections, majority of patients have already developed neutralizing antibodies which can inactivate the virus before it can reach the target cell.

To overcome this problem scientists are currently investigating adenoviruses that infect different species to which humans do not have immunity.

Their primary applications are in gene therapy and vaccination.

Adenoviral genome

The adenovirus genome is a linear, 36 Kb double-stranded DNA (dsDNA) molecule.

It can take up 8kb foreign DNA.

36 kb

Adeno-associated viruses

Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species.

AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.

AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

These features make AAV a very attractive candidate for creating viral vectors for gene therapy.

Applications

Basic research

Viral vectors were originally developed as an alternative to transfection of naked DNA for molecular genetics experiments.

Protein coding genes can be expressed using viral vectors commonly to study the function of the particular protein.

Viral vectors, especially retroviruses, stably expressing marker genes are widely used to permanently label cells to track them and their progeny.

Gene therapy

Gene therapy is a technique for correcting defective genes responsible for disease development. In the future, gene therapy may provide a way to cure genetic disorders.

Diseases result from mutations in the DNA sequence for specific genes, gene therapy trials have used viruses to deliver unmutated copies of these genes to the cells of the patient's body.

Vaccines

Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same as DNA vaccines.

However, since viral vaccines contain only a small fraction of pathogen genes, they are much safer and infection by the pathogen is impossible.

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