Download File - Integrated Science

Introduction to C. elegans
and
RNA interference
Today’s Goals

Review RNAi



How it works to silence genes in C. elegans
How we will use it in the lab
Prepare RNAi
The problem
In order to understand biology, we need
to learn about the function of the
underlying genes
 How can we find out what genes do?
 One way is by eliminating the functional
protein, and examining the phenotype
 Called reverse genetics

Some approaches to Reverse Genetics

Targeted deletion by homologous recombination
(KNOCKOUT MICE) - We’ll discuss next time
 Specific mutational changes can be made
 Time consuming and limited to certain organisms
 Antisense RNA
 Variable effects and mechanism not understood
With the completion of the genome
sequencing project, a quicker, less
expensive reverse genetics method
was needed. Luckily scientists
discovered . . .
RNAi
The old model:
Antisense RNA leads to translational inhibition
mRNA is considered the sense strand
antisense RNA is complementary to the sense
strand
The old model:
Antisense RNA leads to translational inhibition
This can give the same phenotype as a mutant
An experiment showed that the
antisense model didn’t make
sense:

The antisense technology was used in worms

Puzzling results were produced: both sense and
antisense RNA preparations were sufficient to
cause interference.

What could be going on?
1995
Guo S, and Kemphues
KJ.
First noticed that sense RNA was as effective as
antisense RNA for suppressing gene expression
in worm
When researchers looked closely,
they found that double-stranded
RNA caused the silencing!
Negative control
uninjected
Potent and specific
genetic interference by
double-stranded RNA in
Caenorhabditis elegans
Andrew Fire*, SiQun Xu*,
Mary K. Montgomery*,
Steven A. Kostas*†, Samuel E. Driver‡
& Craig C. Mello‡
mex-3B antisense RNA
mex-3B dsRNA
Double-stranded RNA injection reduces the levels of mRNA
1998
Fire et al.
First described RNAi phenomenon in C. elegans by injecting
dsRNA into C. elegans which led to an efficient sequencespecific silencing and coined the term "RNA Interference".
C. elegans is amenable to many
forms of RNAi treament
We are going to inactivate genes by RNAi by feeding
Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)
HOW DOES RNAi WORK?
The Central Dogma
DNA (double-stranded)
RNA (single-stranded)
Protein
When good RNA goes bad....
Viruses can
make doublestranded RNA
When good RNA goes bad....
Cells sense doublestranded RNA and
activate a response
called RNAi
Understanding how
RNAi works is the
key to using it as a
genetic tool and for
therapy
Dicer cuts dsRNA into short RNAs
Vanhecke, D.; Janitz, M. Drug Discov. Today, 2005, 10, 205-225.
Role for a bidentate ribonuclease in the initiation step of RNA interference Emily Bernstein, Amy
A. Caudy, Scott M. Hammond & Gregory J. Hannon
2001
Bernstein
et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and
contains helicase and PAZ domains, as well as two dsRNA-binding
domains.
How does the RNAi that we
are using deliver dsRNA to the
worms?
We are going to feed our worms a
bacterial strain that has been
engineered to express a dsRNA using a
plasmid vector
 Silencinggenomes.org supplies the
bacterial strain with the recombinant
plasmid, already ready to use!

How does the vector induce RNAi?
• Allows E. coli to produce double-stranded RNA
• Requires a specific strain of E. coli
The RNAi feeding vector has two T7
RNA polymerase promoters
• T7 RNA polymerase is a viral polymerase.
• It binds to a specific T7 promoter sequence.
• The L4440 vector is designed with two T7
promoters to make dsRNA.
E. coli strain HT115(DE3) has a modified
lac promoter controlling the
transcription of T7 RNA polymerase
T7 RNA polymerase
T7 RNA polymerase
When IPTG is added T7 RNA
polymerase is expressed this transcribes the dsRNA
T7 RNA
polymerase
Summary
RNAi is a fast and efficient way to
silence gene expression and investigate
gene function
 The RNAi we will perform uses
genetically engineered bacteria that
express dsRNA by induction using IPTG
in the agar, and feeding of the bacteria
to the C. elegans
