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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. SamsonTeaching Assistants: Jenn Cheng and Lisa
Joslin
With additional invaluable help from Lisa Smeester and Rebecca Fry
Monday March 31 DAY 1
Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells
Tues April 1 DAY 1
Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells
Wed April 2 DAY 2
Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data
Thurs April 3 DAY 2
Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data
Monday April 7 DAY 3
Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design new siRNAs for four genes. siRNA is ordered for next experiment
Tues April 8 DAY 3
Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design siRNAs for four genes. siRNA is ordered for next experiment
Wed April 9 DAY 4
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 Informal Presentation of FACS data by students
Thurs April 10 DAY 4
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 Informal Presentation of FACS data by students
Monday April 14 DAY 5
Label isolated RNA and hybridize to microarray slides
Tues April 15 DAY 5
Label isolated RNA and hybridize to microarray slides
Wed April 16 DAY 6
Scan microarray slides and analyze results
Thurs April 17 DAY 6
Scan microarray slides and analyze results
Patriots Day
MIT Holiday
Wed April 23 DAY 7
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 transcipt-
ional 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
Planaria
Trypanosomes Hydra
Protozoa can use RNAi for gene
silencing
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
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!
19 nt duplex
2 nt 3’ overhangs
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!
siRNA
Model for RNAi
processing the dsRNA into 21-23 nt fragments
34
27212016
short-interfering RNA
QuickTime™ and aGIF decompressor
are needed to see this p icture.
step one:
Tuschl, 2001
Dicer contains two RNAse III domains
siRNAs
long dsRNA
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
Tuschl, 2002
the antisense strand of the siRNA guides cleavagestep two:
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
PKR
PP
P
eiF2
dsRNA
Blockage of protein synthesis
interferonproduction
cell deathapoptosis
smaller RNAs can escape the PKR pathway
siRNAs are not recognized by the PKR!
recall that siRNAs are intermediate effectorsIn the RNAi pathway
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?
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