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Vectors - Carriers of Genetic Material
#1
In order to manipulate with genetic material, it is often necessary to have certain carriers of genetic material which would allow its incorporation into the cell.Vectors are the carriers that we use when inserting genetic material into the cell. A great number of different vectors and various methods for entering the gene into the cells have been developed. We distinguish between viral and non-viral vectors:

Viral vectors

Most frequently used viral vectors are those based on adenoviruses, retroviruses, lentiviruses, adeno-associated viruses or herpes simplex virus.

Retroviruses: Most protocols for gene transfer use retroviral vectors. For use in oncology, the ability of retroviruses to integrate into the dividing cells, showed to be an advantage. Attempts of ex vivo transfer proved to be very successful. However, there are several drawbacks. Retroviruses have small genetic capacity of only eight kilobases and serum complement can inactivate them. Currently achieved titer is low in comparison with that that should be achieved for treatment of large tumors. A patient may receive only the limited amount of viruses.

Adenoviruses: Limitations when working with retrovirus imposed the need to find other vectors that could be used with more success in gene therapy. Attention is focused on adenoviruses, as it was revealed that they possess dual DNA so more effective transduction of different cell types is possible regardless of the mitotic stage of the cells. These vectors can be produced in higher titers than retroviruses. Studies have shown that with a minimal amount of viruses, a sufficient level of expression in most tissue scan be achieved, except in haematopoietic cells. Application of the adenoviral vector was first tested in the treatment of nontumorous disorders, such as cystic fibrosis, but it is also in use in cancer therapy. For example, it is in the process of exploring whether there is the possibility of their use when inserting the gene for HSV-TK in patients with tumors of brain and liver, and tumor-suppressor p53 gene in patients with tumors of lungs, head and neck. Although adenoviruses showed good properties for use in gene therapy, they are not without drawbacks. The presence of unmodified viral genes in recombinant virus can trigger an immune response to these antigens, and consequently against the stations that carry it. So far, a great number of viruses with unique properties useful for applications in gene therapy has been explored. Some of them are herpes simplex virus, Avipox virus, Vaccinia virus and Baculovirus.

Non-viral vectors and the "naked" DNA

One of the most promising areas of research is searching for non-viral vectors. They allow the transfer of therapeutic genes into cells without using viruses. In this group belong primarily liposomes, molecular conjugates, and "naked" DNA. Liposomes are combined with the DNA of any size and form lipid-DNA complex, which can be inserted into different cell types. This system, however, has no tissue specificity and therapeutic effectiveness of its use is not yet satisfactory. Because of the need for greater specificity for certain types of cells, molecular conjugates are also used. Molecular conjugates are produced as protein or synthetic molecules with the ability to bind to cell DNS or RNA, on which the desired gene is attached is produced as a protein or a synthetic molecule with the ability to bind to cellular DNA or RNA, in order to make protein-DNA complex. By producing of conjugates, the desired specificity for certain cells is increased, and the disadvantage of this system is too short lifespan of the conjugates. Application of "naked" DNA is the simplest way of its incorporation into target cells without use of viral and non-viral vectors. The station is introduced by mechanical methods such as direct injection into the tissue or bombarding tissues rapidly with DNA bound to gold particles. This method has already been tested in transgenic immunotherapy in the treatment of colon cancer and melanoma. A disadvantage of this method is also the lack of tissue specificity and the need for surgical access of tumor tissue if it is not located in an accessible place.

Gene therapy is not to replace conventional forms of cancer treatment, but, in combination with conventional treatments, it will probably guarantee the improvement of its achievement and, more importantly, will have its use in suppressing residual disease, which occupies the second place on the list of causes of death in humans.
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#2
A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host and into which the DNA fragment to be cloned (called DNA insert) is integrated for cloning. Therefore, a vector must have an origin of DNA replication (denoted as Ori) that functions in the host cell. Any extra-chromosomal small genome e.g., plasmid, phage or virus, may be used as a vector.

Properties of a Good Vector

A good vector must have the following properties:

1. It should be able to replicate autonomously. When the objective of cloning is to obtain a large number of copies of the DNA insert, the vector replication must be under relaxed control that it can generate multiple copies of itself in a single host cell.

2. A vector should ideally be less than 10 kb in size because large DNA molecules are broken during purification procedure. In addition, large vectors present difficulties during various manipulations required for gene cloning.

3. The vector should be easy to isolate and purify.

4. It should be easily introduced into the host cells, i.e., transformation of the host with the vector should be easy.

5. The vector should have suitable marker genes that allow easy detection and/or selection of the transformed host cells.

6. When the objective is gene transfer, it should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.

7. The cells transformed with such vector molecules that contain the DNA insert (recombinant DNA) should be identifiable or selectable from those transformed by the vector molecule only.

8. A vector should contain unique target sites for as many restriction enzymes as possible into which the DNA insert can be integrated without disrupting an essential function.

9. When the expression of the DNA insert is desired, the vector should contain at least suitable control elements, e.g., promoter, operator, and ribosome binding sites; several other features may also be important.
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