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 3Day 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

3427212016

short-interfering RNA

QuickTime™ and aGIF decompressorare needed to see this picture.

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

PeiF2

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?