Gene silencing

Epigenetic regulation of gene expression.

1. Gene silencing Research methods

2. what is gene silencing ? “turning off” a gene this process happens naturally occurs at a transcriptional or a post transcriptional level epigenetic regulation of gene expression.

3. History and Discovery 1. Rich Jorgensen et al. in an attempt to alter flower colours in petunias, introduced additional copies of a gene encoding chalcone synthase, key enzyme for flower pigmentation,into flowers of normally pink or violet colour. 2. Quelling was observed in fungus, Neurospors crassa, in an attempt to boost production of orange pigment produced by the gene aL1 of the fungus. 3. Plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon.

4. Types 1. Transcriptional 2. Post-transcriptional 3. Meiotic

5. 1 . Transcriptional gene silencing (TGS) It Causes gene silencing by: • DNA methylation. • Heterochromatin formation. • Programmed DNA elimination.

6. 2 . Post transcriptional gene silencing It is known commonly as RNA interference (RNAi). It causes silencing by destruction of the mRNA of the gene to which the siRNA shows perfect complementarity.

7. cellular components of gene silencing MicroRNAs (miRNAs) small interference RNAs (siRNAs) Dicer RISC Histones Chromatin and Heterochromatin Transposons .

8. Antisense oligonucleotides Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson.[5][6] These molecules can be composed of single-stranded DNA or RNA and are generally 13–25 nucleotides long. The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism.

9. Ribozymes Ribozymes are catalytic RNA molecules used to inhibit gene expression. These molecules work by cleaving mRNA molecules, essentially silencing the genes that produced them. Sidney Altman and Thomas Cech first discovered catalytic RNA molecules, RNase P and group II intron ribozymes, in 1989 and won the Nobel Prize for their discovery. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes.

10. RNA interference RNA interference (RNAi) is a natural process used by cells to regulate gene expression. It was discovered in 1998 by Andrew Fire and Craig Mello, who won the Nobel Prize for their discovery in 2006.[12] The process to silence genes first begins with the entrance of a double-stranded RNA (dsRNA) molecule into the cell, which triggers the RNAi pathway. The double-stranded molecule is then cut into small double-stranded fragments by an enzyme called Dicer.

11. Applications Medical research Gene silencing techniques have been widely used by researchers to study genes associated with disorders. These disorders include cancer, infectious diseases, respiratory diseases, and neurodegenerative disorders. Gene silencing is also currently being used in drug discovery efforts, such as synthetic lethality, high-throughput screening, and miniaturized RNAi screens.

12. Cancer RNA interference has been used to silence genes associated with several cancers. In in vitro studies of chronic myelogenous leukemia (CML), siRNA was used to cleave the fusion protein, BCR-ABL, which prevents the drug Gleevec (imatinib) from binding to the cancer cells. Cleaving the fusion protein reduced the amount of transformed hematopoietic cells that spread throughout the body by increasing the sensitivity of the cells to the drug.

13. Infectious disease Viruses Viral genes and host genes that are required for viruses to replicate or enter the cell, or that play an important role in the life cycle of the virus are often targeted by antiviral therapies. RNAi has been used to target genes in several viral diseases, such as the human immunodeficiency virus (HIV) and hepatitis. This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells.

14. Bacteria Unlike viruses, bacteria are not as susceptible to silencing by siRNA.[52] This is largely due to how bacteria replicate. Bacteria replicate outside of the host cell and do not contain the necessary machinery for RNAi to function. However, bacterial infections can still be suppressed by siRNA by targeting the host genes that are involved in the immune response caused by the infection or by targeting the host genes involved in mediating the entry of bacteria into cells.

15. Respiratory diseases Ribozymes, antisense oligonucleotides, and more recently RNAi have been used to target mRNA molecules involved in asthma.[53][56] These experiments have suggested that siRNA may be used to combat other respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. COPD is characterized by goblet cell hyperplasia and mucus hypersecretion.[57] Mucus secretion was found to be reduced when the transforming growth factor (TGF)-α was targeted by siRNA in NCI-H292 human airway epithelial cells.

16. Neurodegenerative disorders Huntington's disease Huntington's disease (HD) results from a mutation in the huntingtin gene that causes an excess of CAG repeats.[63] The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus.[64] This disease is incurable and known to cause motor, cognitive, and behavioral deficits. Gene silencing can be used to treat HD by targeting the mutant huntingtin protein. The mutant huntingtin protein has been targeted through gene silencing that is allele specific using allele specific oligonucleotides.

17. Amyotrophic lateral sclero sis Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a motor neuron disease that affects the brain and spinal cord. The disease causes motor neurons to degenerate, which eventually leads to their death. Gene silencing has been used to knock down the SOD1 mutant that is characteristic of ALS.[67][68] In specific, siRNA molecules have been successfully used to target the SOD1 mutant gene and reduce its expression through allele-specific gene silencing.

18. Therapeutics challenges There are several challenges associated with gene silencing therapies, including delivery and specificity. For instance, in the case of neurodegenerative disorders, gene silencing molecules must be delivered to the brain. The blood-brain barrier makes it difficult to deliver molecules into the brain by preventing the passage of the majority of molecules that are injected or absorbed into the blood.Thus, researchers have found that they must directly inject the molecules or implant pumps that push them into the brain.

19. Food Arctic Apples are a suite of trademarked[70] apples that contain a nonbrowning trait create by using gene silencing to reduce the expression of polyphenol oxidase (PPO) It is the first approved food product to use that technique.

20. PRESENTED BY : V. VIGNESH