Gene splicing

1. A SEMINAR PRESENTATION
ON
GENE SILENCING
(SESSION: 2022- 2023)
SUBMITTED BY SUBMITTED TO
ANKITA PAL DEPARTMENT OF BIOTECHNOLOGY
ROLL NO : 191411029007 IET, BUNDELKHAND UNIVERSITY,
B.TECH (BIOTECHNOLOGY) JHANSI, UTTARPRADESH-284128

2. CONTENTS
1) Introduction.
2) Types of gene silencing.
3) Research Methods.
4) Applications.
5) Conclusion.
6) Reference.

3. INTRODUCTION
GENE SILENCING:-
 Gene silencing is the “regulation of the gene expression” in a cell to prevent the expression of a certain
gene. Gene silencing can occur during either transcription or translation.
 The method of genes silencing is used to produce therapeutics to combat cancer and some diseases, like
infectious diseases and neurodegenerative disorders.
 Gene silencing is often considered the same as “gene knockdown”, because the methods used to silence
genes, such as RNAi, siRNA, etc generally reduce the expression of a gene by at least 70% but do not
completely eliminate it.
 Gene silencing methods are often considered better than gene knockdowns, since they allow
researchers to study essential genes that are required for the animal models to survive and cannot be
removed.

4. TYPES OF GENE SILENCING
 There are methods of gene silencing, and they are as follows:-
1. Transcriptional gene silencing.
2. Post- transcriptional gene silencing.
3. Meiotic gene silencing.
1) Transcriptional Gene Silencing:-
 Transcription gene obscures transcription via the methylation of 5’ untranslated region (5’UTR).
 Transcriptional gene silencing is mechanistically distinct from the RNA interference gene silencing pathway.
 This method can result in long- term stable epigenetic modifications to gene expression that can be passed on
to a daughter cells during cell- division, whereas RNAi does not.
 Types are:-
i. Genomic imprinting.
ii. Transgene silencing.
iii. Position effect.
iv. RNA- directed DNA methylation.

5. 2) Post-Transcriptional Gene Silencing:-
 In this method, gene silencing exploits the cellular mechanism where in transcripts having sequence
similarity to the double- stranded RNA (ds-RNA) molecules present in the cell will be subjected to
degradation.
 This method is closely related to natural processes such as “RNA- mediated virus resistance” and “cross-
protection in plants”.
 Post- transcription gene silencing has shown great potential in silencing the deleterious genes efficiently,
so that value- added plant products could be obtained.
 Types are:-
i. RNA silencing.
ii. RNA interference.
iii. Nonsense mediated decay.

6. 3) Meiotic Gene Silencing:-
 It is a term use to describe “mega base- scale chromatin remodelling process” in which genes are
located along un- synapsed chromosomes are transcriptionally inactivated during meiotic prophase- 1.
 This phenomenon was described in filamentous fungi.
 The silencing of gene expression by segments of DNA present in excess of the normal is called
suppression in plants and quelling in fungi.
 There is a related process called as meiotic silencing by unpaired DNA, in which DNA unpaired in
meiosis causes silencing of all DNA homologous to it , including genes that are themselves paired.
 Types are :-
i. Transvection.
ii. Meiotic silencing of unpaired DNA.

7. RESEARCH METHODS:-
1) Antisense oligonucleotides:-
 Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson.
 Oligonucleotides which are short nucleic acid fragments bind to complementary target mRNA
molecules when added to the cell.
 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:-
a) By using a RNase H- dependent mechanism.
b) By using a steric blocking mechanism.
 RNase H- dependent oligonucleotides cause the target mRNA molecules to be degraded, whereas
steric- blocker oligonucleotides prevent translation of the mRNA molecule.

8. 2) RNA interference:-
 RNA interference is a natural process used by cells to regulate gene expression. It was discovered by
Andrew Fire and Craig Mello in 1998, also won Nobel Prize for this discovery.
 The process to silence genes first begins with the entrance of a double- stranded RNA(ds- RNA) molecule
into the cell, which triggers the RNAi pathway.
 When RNAi pathway is triggered, then the double- stranded molecule is cut into small double stranded
fragments by an enzyme called dicer. These small fragments include micro RNA (miRNA) and small
interfering molecule (siRNA), and are of approximately 21-23 nucleotides in length.
 The genes can be silenced by siRNA molecules that cause the endonucleatic cleavage of the target
mRNA molecules or by miRNA molecules that suppress translation of the mRNA molecule. With the
cleavage or translational repression of the mRNA molecules, the genes that form them are rendered
essentially inactive.
 RNAi is thought to have evolved as a cellular defence mechanism against invaders, such as RNA viruses,
or to combat the proliferation of transposons within a cell’s DNA.

9. APPLICATIONS:-
 Gene silencing techniques have been widely used by researchers to study genes associated with
disorders, and these disorders include cancer, respiratory diseases, Huntington’s disease, etc.
 Gene silencing is also currently being used in drug discovery efforts, such as synthetic lethality, high-
throughput screening and miniaturized RNAi screens.
 Some application includes are:-
i. CANCER:-
 RNA interference has been widely used to silence genes associated with several cancers.
 In in- vitro studies of chronic myelogenous leukaemia (CML), siRNA was used to cleave the fusion
protein, BCR-ABL, which prevents the drug Gleevec (imatinib) from binding to the cancers.
 RNA interference can also be used to target specific mutants. For E.g. siRNAs were able to bind to
specifically to tumour suppressor protein p53 molecules containing a single point mutation and
destroy it, while leaving the wild- type suppressor intact.

10. ii. RESPIRATORY DISEASES:-
 Antisense oligonucleotides, and more recently RNAi have been used to target mRNA molecules involved in
asthma.
 The experiments have suggested that siRNA may be used to combat to other respiratory diseases such as,
chronic obstructive pulmonary diseases (COPD) and cystic fibrosis.
 COPD is characterized by “goblet cell hyperplasia and mucus hypersecretion”. Mucus secretion was found
to be reduced when the transforming growth factor- alpha was targeted by siRNA in NCL-H292 human
airway epithelial cells.
iii. HUNTINGTON’S DISEASE:-
 Huntington’s disease result from mutation in the huntingtin gene that causes an excess of CAG repeats.
The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus.
 Gene silencing can be used to treat Huntington’s disease by targeting the mutant huntingtin protein. In
this, mutant huntingtin protein has been targeted through gene silencing is allele specific i.e. “using allele
specific oligonucleotides”.
 In this method, the antisense oligonucleotides are used to target single nucleotide polymorphism (SNP),
which are single nucleotide changes in the DNA sequence, since Huntington's disease patients have ben
found to share common SNPs that are associated with the mutated huntingtin allele.

11. CONCLUSION
Gene silencing effects especially post- transcriptional effects were first observed in plants or fungi, but not
yet have been reported from studies of mammals. But it does not mean that gene silencing is weaker or non-
existent in mammals. It is striking that post- transcription gene silencing was observed in a wide range of
taxa before its documentation in a mammal , despite the fact that the observed majority of gene expression
studies are conducted on mammals.
It has been noted that host- mediated gene –silencing pathways could be used to extinguish the expression
of harmful genes. This approach has many of the virtues of a vaccine in the sense that an existing host
defence system is stimulated, and other approaches to specific downregulation do not enjoy this advantage.
On the other hand, gene silencing has already comprised a number of gene transfer efforts and is likely to
represent a barrier to many of the forms of gene therapy currently under development.

12. REFERENCES
1) Taken from web sources.
2) Gene silencing. National centre for Biotechnology Information. Retrieved 11 November 2013.
3) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC289175/