1. Presented by
SK SARUK ISLAM
M.Sc( 2ND SEM), 2016
ROLL- VU/PG/MCB IIS, NO- 09
Reg.No- 212871 0f 2012-13
Vidyasagar University
Dept. of Microbiology
2. Introduction
Discovery of shRNA
Why study about shRNA
Goal
Vector choice for expression shRNA
Invention of shRNA
Mechanism of action
shRNA can impair and cell function and survival
Application
What’s new about shRNA
In summary
References
3. A small hairpin RNA or short hairpin RNA ( shRNA ) is an artificial RNA molecule with a tight
hairpin turn that can be used to silence target gene expression via RNA
interference (RNAi).
RNA interference (RNAi) is a mechanism where the presence of certain fragments of ds RNA
interfieres with the expression of a particular gene which shares a homologous
sequence with this dsRNA.
Expression of shRNA in cells is typically accomplished by delivery of plasmids or
through viral or bacterial vectors.
ShRNA is an advantageous mediator of RNAi in that it has a relatively low rate of
.
The siRNAs and miRNAs are derived from the processing of endogenously encoded short
hairpin RNAs (shRNAs). In contrast, the exogenous siRNAs hypothetically represent perfect
drugs for the specific blocking of unwanted or disease-causing gene products .
4. • In 1990 why the flowers color are more vivid in plant
• 1995 injected the antisense strand RNAs of certain endogenous gene into
worms that may hybridized to corresponding gene & block
translation.
• In 1998, Fire & Mello found that dsRNA was at least 10 fold or perhaps a
100 fold more potent as a silence trigger than sense /antisense alone.
• Escherichia coli , containing a plasmid with shRNA, fed to mice can
knock-down target gene expression in the intestinal epithelium.
• In 2012 try to treat patients with Familial Adenomatous
Polyposis.
1990 1995-1998 2012
5. • Breast Cancer, second most
common form of cancer in
women, behind skin cancer.
• 72,007 men and 69,398 women
were diagnosed with
colorectal cancer & 26,781
men and 26,224 women died from
colorectal cancer.
ShRN
A
Analy
sis
Those
Canc
er
Cells
7. Vector Choice for Expression Of
shRNA
Vector Element Utility
5' LTR Necessary for lentiviral particle production and integration into host cell genome
Ψ Viral genome packaging sequence using lentiviral packaging systems
RRE Enhances titer by increasing packaging efficiency of full-length viral genomes
tGFP or tRFP TurboGFP or RFP reporter for visual tracking of transduction and expression
None No reporter option for use in applications where fluorescence is not required
Puro R Permits antibiotic-selective pressure and propagation of stable integrants
Scaffold microRNA-adapted shRNA for gene knockdown
WPRE Enhances transgene expression in the target cells
3' SIN LTR 3' Self-inactivating Long Terminal Repeat for increased lentivirus safety
8. .
Invention of shRNA
shRNA
► Once the vector has
integrated into the host
genome, the shRNA is then
transcribed in the
nucleus by polymerase
II or polymerase III
depending on the
promoter choice
► The primary transcripts
are processed by the
Drosha or DGCR8
complex and form pre-
shRNAs
►Pre-shRNAs are
transported to the
cytoplasm via
9. ① Endogenous Exportin 5 for nuclear
export
② Ago2 is recruited by TRBP , that
forms a dimer with Dicer & receives the
shRNA .
③shRNA cleaved by Dicer generating a
19-23 nt duplex siRNA with 2 nt 3’
overhangs.
④ Identify the “guide strand” &
cleaved the “passenger strand” by
Ago2 .
⑤ The “passenger strand” is released.
⑥ “guide strand” is integrated in the active
RISC that contains argonaute-associated
proteins .
⑦ The siRNA guides RISC to the target
mRNA.
⑧ RISC delivers the mRNA to cytoplasmic
foci i.e P-bodies/GW-bodies.
10. shRNA Can Impair Cell Function And
Survival
►RISC is a
ribonucleoprotei
n
►In perfect
complementarity ,
RISC degrade the
mRNA
► But in imperfect
complementarity ,
RISC represses
translation of the
mRNA
►In the both of cases ,
the shRNA leads to
11. shRNA offers the possibility of prolonged gene silencing.
Viral based shRNAs have also been used to evaluate gene
function at whole genome scale using pool of silencing construct.
Pooled or arrayed screens are now widely used to perform high-
throughput screens to identify genes required in a Varity of
processes such as :
►Nasopharyngeal Carcinoma
►Head and Neck Cancer
►Oral Squamous Cell Carcinoma
►Tooth Development
►Regulation of Splice Variants
►Neurodegenerative Disorders
12. Gradalis, Inc. developed the FANG vaccine, which is used in
treatment of advanced cancers.
Marina Biotech developed CEQ508 which is used to treat
Familial Adenomatous Polyposis. CEQ508 uses a
bacterial vector to deliver shRNA against β-catenin.
Gradalis, Inc. developed bi-functional shRNA STMN1, which is
used to treat advanced and/or metastatic cancers, against
stathmin 1 and is delivered intratumorally through bi-
lamellar invaginated vesicle (BIV) lipoplex (LP)
technology.
13. In summary…
It is now possible to easily
identify specific anti-
proliferative genes for
numerous cancer cell
lines.
14. Chendrimada TP. et al., TRBP recruits the Dicer complex to Ago2 for microRNA processing and
gene silencing. Nature,2005. 436(7051):p.740-744.
Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis
elegans. Nature,1998. 391(6669): p. 806-811.
Guo, S. and K.J. Kemphues, par-1, a gene required for establishing polarity in C. elegans embryos,
encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995. 81(4): p. 611-620.
Macrae I, Zhou K, Li F, Repic A, Brooks A, Cande W, Adams P, Doudna J. "Structural basis for
doublestranded RNA processing by dicer". Science ,2006.311 (5758):p.195–198.
Stephanie E.Mohr., et ai., RNAi screening comes of age : improved techniques and
complementary approaches. Nature ,2014. 15:p.591-600.
Wang, Zhaohui; Rao, Donald D.; Senzer, Neil; Nemunaitis, John . "RNA Interference and Cancer
Therapy". Pharmaceutical Research,2011.28 (12): p.2983–2995.
Xiang, Shuanglin; Fruehauf, Johannes; Li, Chiang J . "Short hairpin RNA–expressing bacteria elicit
RNA interference in mammals". Nature Biotechnology ,2006.24 (6):p. 697–702.
