Transcript Document

BEH.109: Laboratory Fundamentals in Biological
Engineering.
MODULE 3
Eukaryotic Cells as Phenotypic
Indicators:
The use of RNAi to modulate gene
expression
Instructor: Leona D. Samson
Teaching Assistants: Jenn Cheng and Lisa
Joslin
With additional invaluable help from Lisa
Smeester and Rebecca Fry
Monday March 31
DAY 1
Tues April 1
DAY 1
Wed April 2
DAY 2
Thurs April 3
DAY 2
Module 3 Overview &
mini-lecture on RNAi
Module 3 Overview &
mini-lecture on RNAi
Comprehensive lecture
on RNAi with some
examples
Comprehensive lecture
on RNAi with some
examples
Safety Orientation
Safety Orientation
Sterile Technique
Sterile Technique
Transfection of EGFP &
p53 siRNA into EGFP
expressing HeLa cells
Transfection of EGFP &
p53 siRNA into EGFP
expressing HeLa cells
Harvest transfected cells
Microscope analysis &
FACS analysis
Analyze data
Harvest transfected cells
Microscope analysis &
FACS analysis
Analyze data
Monday April 7
DAY 3
Tues April 8
DAY 3
Wed April 9
DAY 4
Thurs April 10
DAY 4
Introduction to the
ATM, ATR, EXO1 and
AAG genes
Introduction to the
ATM, ATR, EXO1 and
AAG genes
Ambion and Blast
session to design new
siRNAs for four genes.
Ambion and Blast
session to design
siRNAs for four genes.
siRNA is ordered for
next experiment
siRNA is ordered for
next experiment
Monday April 14
DAY 5
Tues April 15
DAY 5
Label isolated RNA and
hybridize to microarray
slides
Label isolated RNA and
hybridize to microarray
slides
Introduction to DNA
microarrays and
overview of what will
happen on days 5 & 6
Introduction to DNA
microarrays and
overview of what will
happen on days 5 & 6
Transfect four new
si.RNAs; cellular RNA
will be isolated over the
w/e
Transfect four new
si.RNAs; cellular RNA
will be isolated over the
w/e
Informal Presentation of
FACS data by students
Wed April 16
DAY 6
Informal Presentation of
FACS data by students
Thurs April 17
DAY 6
Scan microarray slides
and analyze results
Scan microarray slides
and analyze results
Wed April 23
DAY 7
Patriots Day
MIT Holiday
MODULE 3 Student
Presentations
Thurs April 24
DAY 7
MODULE 3 Student
Presentations
Snapshot of
the next four
weeks
We will
eliminate the
expression of
six different
genes using
RNAi
technology,
human cells,
fluorescent
proteins and
DNA
microarrays
You WILL be required to write a lab
report, but Class still happens on
April 24rd.
Please follow the "BEH.109
Guidelines for Module Reports"
handout that was given to you
previously.
The report will be DUE ON
MONDAY, APRIL 29th BY 5 PM.
RNA Interference - RNAi
BE109 Module 3
Day 2 lecture
RNA interference first
discovered in Petunias!
Called PTGS, for “Post
Transcriptional Gene
Silencing”
Color
changes
can be
induced
by RNAi,
or PTGS..
Post
transciptional
gene
silencing
Small (21-23 nts) RNA duplexes, with the
same sequence as in the silenced gene, were
identified as being responsible for knocking
down expression
So what other organisms can
do this thing called PTGS?
“Post Transcriptional Gene
Silencing”
Arabidopsis thaliana
Protozoa can use
RNAi for gene
silencing
Planaria
Trypanosomes
Hydra
RNAi can
operate in
insects
too!
Sulston and Horvitz (1977). Develop. Biol. 56, 110-156.
RNAi is used
by C. elegans
to control the
timing of
development of
various tissues
Such gene
silencing is a
natural
phenomenon
in this
organism
19 nt duplex
2 nt 3’ overhangs
This dsRNA species found in plants, C.
elegans and Drosophila melanogaster
undergoing gene silencing….but how to prove
it is responsible?
Purified them and showed in vitro silencing in
Drosophila extracts; used sythetic sdRNA
oligo to achieve same thing!
C. Elegans movie
C. Elegans grow
on agar dishes
with E. coli
bacteria as a
source of food.
If they eat E. coli
expressing
dsRNA
molecules…this
creates specific
knock-down of
gene expression!
In C. elegans the
siRNA effects
can be amplified
making the
silencing quite
stable
This does not
appear to
happen in
mammalian cells
(RISC = RNAi
Induced Silencing
Complex; RdRP =
RNA dependent
RNA polymerase)
http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v421/n6920/full/421220a_fs.html&content_filetype=pdf
19,757 genes
16,757 have
been
inactivated by
RNAi
10% display
spontaneous
phenotype;
this 10% is
enriched for
conserved
genes
19,757 genes
16,757 knock
down mutants
were screened
for body fat
content
305 knock
downs had
increased body
fat, 112 genes
had
decreased..new
targets for
obesity?
C. Elegans movie
So…what about RNAi in
mammalian cells…
May 2001…the first report…
How does RNAi work
in mammalian cells?
RNAi works postranscriptionally……..
in key two steps!
Model for RNAi
siRNA
step one:
processing the dsRNA into 21-23 nt fragments
QuickT ime ™an d a
GIF deco mpre ssor
ar e need ed to see this pictur e.
34
27
21
20
16
short-interfering RNA
Tuschl, 2001
Dicer contains two RNAse III domains
long dsRNA
siRNAs
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
step two:
the antisense strand of the siRNA guides cleavage
Tuschl, 2002
RNAi silencing complex
• may be associated with translating ribosomes
• active RNAse enzyme not yet identified
• may participate in endogenous pathways that
silence genes via translational repression
Mammals exhibit potent responses to dsRNA
P P
dsRNA
PKR
interferon
production
P
eiF2a
apoptosis
Blockage of protein synthesis
cell
death
smaller RNAs can escape the PKR pathway
recall that siRNAs are
intermediate effectors
In the RNAi pathway
siRNAs are not recognized
by the PKR!
Two ways to get double stranded RNA
lentiviral construct for siRNAs
Rubinson et al Nature
Genetics, 2003
Practical Aspects of RNAi
• biological research
– defining gene function (gene knockout)
• C. elegans genome RNAi projects
– defining biochemical pathways
• microarray screening of RNAi knockouts
• therapeutic treatment
– cancer
– viral infection
– parasitic infection
HIV levels
can be
reduced by
30-50 fold by
siRNA!!!
What is the killer
virus that might
sweep the
globe?
Will RNAi
eventually
become an
effective and
specific antiviral
therapy?