2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. Adenoviral genome
The adenovirus genome is a linear, 36 Kb double-stranded DNA (dsDNA)
molecule.
It can take up 8kb foreign DNA.
36 kb
10.
11. 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.
12. 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.
13. Gene therapy
Gene therapy is a technique for correcting defective genes responsible
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
genes to the cells of the patient's body.
14. 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.
