Gene knockoff

“GENE KNOCKOFF” by RNAi
Technology
TANVI
Ph.D I
MBB 605

 Gene silencing is a technique that aims to reduce or eliminate
the production of a protein from it’s corresponding gene.
 It generally describe the “switching off” of a gene by a
mechanism other than genetic modification i.e., a gene which
would be expressed (“turned on”) under normal circumstances
is switched off by machinery in the cell.
 It occurs when RNA is unable to make a protein during
translation.
 Gene silencing is same as gene knock off but is totally different
from gene knock out.
 When genes are knock off ,there expression is reduced , where
in contrast when genes are knocked out, they are completely
erased from organism’s genome and thus have no expression.
INTRODUCTION

1990 Jorgensen:
 To deepen the pigmentation in petunias, introduction of transgenes
homologous to endogenous genes often resulted in plants with both
gene suppressed called co suppression.
 Resulted in degradation of the endogenous and transgene mRNA.
1995 Guo and Kemphues:
 injection of either antisense or sense RNAs in the germline of C.
elegans was equally effective at silencing at homologous target
genes.
1998 Mello and Fire:
 Extension of above experiments, combination of sense and antisense
RNA(=dsRNA) was 10 times more effective than single strand RNA.
Short History of gene silencing

 The discovery of the mechanism of RNA interference by ds RNA by
Prof. Andrew Fire and Prof. Craig Mello in 1998, gave them the Nobel
prize in 2006.
2001 Tuschl T and colleagues
 First described RNAi in mammalian cells
2003 Song E
First reported that siRNAs can be used therapeutically in whole animals
HOW does it works?
1. This is accomplished by binding a specific strand of RNA to an
existing m-RNA strand.
2. The m-RNA creates a copy of DNA strand.
3. By binding the RNA to the m-RNA, m-RNA is prevented from
replicating that portion of the DNA.
4. Specific genes can be targeted and prevented from replicating in to
new DNA strands.

Gene silencing methods used in
research
Antisense oligonucleotides
 discovered by Paul
Zamecnik and Mary
Stephenson in 1978
 Oligonucleotides are
short nucleic
acid fragments, that
bind to
complementary
target mRNA
molecules when added
to the cell.
Ribozymes
• Sidney Altman
and Thomas Cech first
discovered catalytic
RNA molecules
• These are catalytic RNA
molecules used to inhibit
gene expression.
• cleaves mRNA molecules,
essentially silencing the
genes that produced
them.
RNAi

Transcriptional Post transcriptional
Types of gene silencing
 Genome imprinting
 Paramutations
 RNA directed DNA
methylation
 Transposon silencing(Histone
modification)
 Transgene silencing
 Position effect
 RNA Interference
 RNA silencing
 Non sense mediated
decay

 RNA interference ( RNAi), is a technique in which exogenous, double-stranded RNAs
( dsRNAs ) that are complimentary to known mRNAs, are introduced into a cell to
specifically destroy that particular mRNA, thereby diminishing or abolishing gene
expression.
 RNA interference was known by other names, including post transcriptional gene
silencing (Plants)and quelling(Fungi) .
RNAi mediated gene knockoff
RNA Interference

Defense Mechanism
 Defense against Infection by viruses.
 As a defense mechanism to protect against transposons
and other insertional elements
Genome Wide Regulation
 RNAi plays a role in regulating development and
genome maintenance.
 30% of human genome regulated
Need For Interference

RNAi was found to work in many
diverse species
Fungi
Trypanosomes
Insects
Zebrafish
Mice

Provides valuable insight into evolution
 found in all eukaryotes except some fungi= ancient biochemical
mechanism
 “indicates that gene expression is the key to evolving complex
organisms”
Invaluable tool for functional genomics
 Knockdowns are easier than knockouts= saves time and money
 " Allows for study of function with a variety of controls:
positive, negative, rescue”
Why RNAi is important

RNAi pathway guided by,
Si RNA (small interfering RNA):
 Small interfering RNAs that have an integral role in the
phenomenon of RNA interference (RNA i), a form of post-
transcriptional gene silencing
 RNAi: 21-25 nt. fragments, which bind to the complementary
portion of the target mRNA and tag it for degradation
Mi RNA (micro RNA):
 mi RNA Originate from capped & polyadenylated full length
precursors (pri-miRNA)
 Hairpin precursor ~70 nt (pre-mi RNA) Mature mi RNA ~22 nt (mi
RNA)
 mi RNA originates from ssRNA that forms a hairpin secondary
structure.
 Mi RNA regulates post-transcriptional gene expression.

Dicer:
 RNAse III-like dsRNA-specific ribonuclease
• Enzyme involved in the initiation of RNA i.
• It is able to digest dsRNA into uniformly sized small RNAs (si
RNA)
 Dicer family proteins are ATP- dependent nucleases.
 Loss of dicer→loss of silencing process in vitro
RISC (RNA Inducing Silencing Complex):
 RISC is a large (~500-kDa) RNA-multi protein complex, which
triggers mRNA degradation in response to Si RNA
 Unwinding of double- stranded Si RNA by ATP independent
helicase.
 The active components of an RISC are endonucleases called
argonaute proteins which cleave the target mRNA strand.

 The long dsRNAs enter a cellular pathway that is
commonly referred to as the RNA interference
(RNAi) pathway.
 First, the dsRNAs get processed into 20-25
nucleotide (nt) small interfering RNAs (siRNAs)
by an RNase III-like enzyme called Dicer.
 Then, the siRNAs assemble into
endoribonuclease-containing complexes known
as RNA-induced silencing complexes (RISCs),
unwinding in the process.
 The siRNA strands subsequently guide the
RISCs to complementary RNA molecules, where
they cleave and destroy the cognate RNA (
 Cleavage of cognate RNA takes place near the
middle of the region bound by the siRNA strand.
The Mechanism of RNA Interference

RNA interference has been used for applications in biotechnology,
particularly in the engineering of food plants that produce lower levels
of natural plant toxins.
For example, cotton seeds are rich in dietary protein but naturally
contain the toxic terpenoid product gossypol, making them unsuitable
for human consumption.
 RNAi has been used to produce cotton stocks whose seeds contain
reduced levels of delta-cadinene synthase, a key enzyme in gossypol
production, without affecting the enzyme's production in other parts of
the plant, where gossypol is important in preventing damage from plant
pests.
Similar efforts have been directed toward the reduction of the
cyanogenic natural product linamarin in cassava plants
Role of RNAi in Biotechnology…

It may be possible to exploit RNA interference in therapy.
A key area of research in the use of RNAi for clinical applications is the
development of a safe delivery method, which to date has involved mainly
viral vector systems similar to those suggested for gene therapy
Potential treatments for neurodegenerative diseases have also been
proposed, with particular attention being paid to the polyglutamine
diseases such as Huntington's disease.
RNA interference is also often seen as a promising way to treat cancer by
silencing genes differentially upregulated in tumor cells or genes involved
in cell division.
Modulation of HIV-I replication by RNA i.
Role of RNAi in Medicine…

Current trends in clinical trials using RNAi
 Many pharmaceutical companies devoted to RNAi research are coming up with
interesting insights into how human ailments can be cured using the RNAi
mechanism. Although the trials are in their early phases. For example, Nucleonics
Inc., a pharmaceutical company, has initiated a phase l clinical trial of the
systemically administrated RNAi-based therapeutic NucB-1000 for the potential
treatment of HBV infection. NucB-1000 consists of a plasmid DNA construct
designed to produce four different shRNAs, targeting different sequences of the
HBV genome, under control of an RNA polymerase III promoter. The plasmid
DNA was formulated with a cationic-lipid delivery system.
 HIV was the first infectious agent targeted by RNAi perhaps owing to the fact that
the life cycle of HIV is well understood as its pattern of gene expression. Synthetic
and expressed siRNAs have been used to target a number of early and late HIV-
encoded RNAs including the TAR element, tat, rev, gag, env, vif, nef and reverse
transcriptase. Cellular cofactors, such as NFkb, the HIV receptor CD4 and co-
receptors CXCR4 and CCR5 have also been successfully down regulated by RNAi
resulting in an inhibition of HIV replication
RNA interference: A futuristic tool and its
therapeutic applications
Anghesom Ambesajir et al, Saudi Journal of Biological Sciences(2012)

Animal models are widely used to investigate the therapeutic
efficiency of RNAi. in vivo utilization of siRNA was effectively
performed by targeting the colorectal cancer-associated gene β-
catenin. Decreased proliferation and diminished invasiveness
were observed following siRNA-mediated silencing of this gene
in human colon cancer cells. Additionally, when treated cancer
cells were placed in a nude mouse, prolonged survival was seen
compared with mice receiving unmanipulated tumors.
The future of RNAi researches is exciting and there are many
applications to be considered in this field. An instance where
RNAi technology can be used to silence the gene(s) responsible
for the production of boxalylaminoalanine-L-alanine (BOAA), a
neurotoxin found in a leafy vegetable known as Lathyrus
sativus, which is commonly used as a food by the people in the
lower socioeconomic class of the poor nations.

The ripening hormone, ethylene is known to initiate, modulate and co-ordinate the expression of
various genes involved in the ripening process. Therefore ethylene is held accountable for the
tons of post-harvest losses due to over-ripening and subsequently resulting in fruit rotting. In
the present investigation, delayed ripening tomatoes were generated by silencing three homologs
of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene during the course of
ripening using RNAi technology. The chimeric RNAi-ACS construct designed to target ACS
homologs, effectively repressed the ethylene production in tomato fruits. Fruits from such lines
exhibited delayed ripening and extended shelf life for∼45 days, with improved juice quality. The
ethylene suppression brought about compositional changes in these fruits by enhancing
polyamine (PA) levels. Further, decreased levels of ethylene in RNAi-ACS fruits has led to the
altered levels of various ripening-speciﬁc transcripts, especially the up-regulation of PA
biosynthesis and ascorbic acid (AsA) metabolism genes and down-regulation of cell wall
hydrolyzing enzyme genes. These results suggest that the down-regulation of ACS homologs
using RNAi can be an effective approach for obtaining delayed ripening with longer shelf life
and an enhanced processing quality of tomato fruits. Also, the chimeric gene fusion can be used
as an effective design for simultaneous silencing of more than one gene. These observations
would be useful in better understanding of the ethylene and PA signalling during fruit ripening
and molecular mechanisms underlying the interaction of these two molecules in affecting fruit
quality traits.
Delayed ripening and improved fruit processing quality in
tomato by RNAi-mediated silencing of three homologs of
1-aminopropane-1-carboxylate synthase gene
Aarti Gupta, Ram Krishna Pal, Manchikatla Venkat Rajam
received:
4
October
2012
Accepted
:
18
February
2013
Publishe
d:16
March
2013

Generation of tomato transformants With RNAi-ACS
(chimera)construct
Isolation of small RNA for detection of siRNA in RNAi-ACS tomato
line
Estmiation of rate of ethylene production in tomato fruits
Polyamine analysis
Determination of fruit shelf life
Determination of ascorbic acid content

Fruits from all thirty RNAi-ACS tomato lines along with unrelated control and WT plants
were analysed for rate of ethylene evolution. Results shows that fruits from RNAi-ACS lines
liberated reduced levels of ethylene. Ethylene liberation was found to be least in RNAi-
ACS60 and RNAi-ACS81, releasing only 4–5% when compared with controls (WT and UR).
Fruits of other RNAi-ACS lines showed 10–70%of ethylene evolution to that of control
fruits. This difference in ethylene evolution among RNAi-ACS lines could be attributed to
variable suppression of the target gene.
On the basis of RT-PCR and siRNA detection results, RNAi-ACS lines were categorized into
highly silenced lines (RNAi-ACS60 and RNAi-ACS81) and moderately silenced lines
(RNAi-ACS123 and RNAi-ACS125).
Reduction in ethylene levels has led to signiﬁcant reduction in CO2 evolution (marker for
respiration) in RNAi-ACS tomato lines over controls (WT and UR). Among the various lines
analyzed For rate of respiration, RNAi-ACS60 and RNAi-ACS81 were found to exhibit
∼50% reduction in respiratory activity in harvested .Fruits over control fruits, while rest of
the RNAi-ACS lines showed up to30% reduction in rate of Respiration. The variation in
respiratory activity of different fruits corresponds to the different levelsof ethylene liberated
by such fruits.
Increased PA accumulation in fruits of RNAi-ACS lines may have Also inﬂuenced the
enhanced shelf life possibly by stabilizing the membranes.
RESULTS

 It is the regulation of gene expression and widely used in agriculture
and biotechnology.
 Besides the all types of gene silencing the RNAi is the important post
transcriptional gene silencing.
 It is an important part of the cellular machinery that provides viral
immunity and a mechanism for the control of gene expression.
 A variety of RNA triggers function in the RNAi mechanism result in
gene suppression that can be both temporary and permanent.
RNAi approaches are economical and provide a tremendous amount
of flexibility by allowing time- and tissue-specific knockdown of genes or
specific splice forms, in addition to avoiding the common problem of
embryonic lethality from gene knockout.
The field is relatively young and much remains to be discovered
Conclusion

Gene knockoff

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