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Biochemistry

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Purines, Pyrimidines and Nucleic Acid Metabolism

Question
29 out of 59
 

Which is not a method of gene therapy? (AIIMS Nov 2010)



A FISH
B Transfection

C Electroporation
D Bacteriophage

Ans. A FISH

a. Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease.

b. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutated gene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection.

c. Although the technology is still in its infancy, it has been used with some success. Scientific breakthroughs continue to move gene therapy toward mainstream medicine.

Types Of Gene Therapy

1. Germ line gene therapy

a. In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are ordinarily integrated into their genomes.

b. The change due to therapy would be heritable and would be passed on to later generations.

c. This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary

diseases.

d. Many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.

2. Somatic gene therapy

In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.

Vectors in gene therapy

1. VIRUSES

a. All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus.

b. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected.

c. Some types of viruses insert their genes into the host's genome, but do not actually enter the cell. Others penetrate the cell membrane disguised as protein molecules and enter the cell.

d. Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. Concerns about the safety of adenovirus vectors were raised after the 1999 death of Jesse Gelsinger while participating in a gene therapy trial.

e. Since then, work using adenovirus vectors has focused on genetically crippled versions of the virus. Now that the genetic material of the virus has been inserted, it can be said that the host cell has been modified to contain new genes.

f. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately.

g. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Britain, Italy and Japan.

h. Herpes Simplex Virus is a human neurotropic virus. This is mostly examined for gene transfer in the nervous system.

2. Non-viral methods

a. Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two.

b. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses

3. Naked DNA

a. This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection.

b. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success.

c. This success, however, does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

d. Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field.

e. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.

f. Sonoporation, or cellular sonication, is the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane.

g. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation.

h. A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information.

i. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.

j. This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus:

k. it can also transform organelles, including plastids The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process.

l. FISH is fluorescent -in-situ hybridization and is a method of detection and not related with gene therapy

m. Fluorescence in situ hybridization (FISH) is a very sensitive technique that is also used for this purpose.

n. This often places the gene at a location on a given band or region on the chromosome.

o. Some of the human genes localized using these techniques are listed..

p. This table represents only a sampling, since thousands of genes have been mapped as a result of the recent sequencing of the human genome.

q. Once the defect is localized to a region of DNA that has the characteristic structure of a gene, a synthetic gene can be constructed and expressed in an appropriate vector and its function can be assessed—or the putative peptide, deduced from the open reading frame in the coding region, can be synthesized.

r. Antibodies directed against this peptide can be used to assess whether this peptide is expressed in normal persons and whether it is absent in those with the genetic syndrome.

Purines, Pyrimidines and Nucleic Acid Metabolism Flashcard List

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