1. Introduction to C. elegans
and
RNA interference

2. Today’s Goals Review RNAi Prepare RNAi
How it works to silence genes in C. elegans
How we will use it in the lab
Prepare RNAi

3. 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

4. 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

5. With the completion of the genome sequencing project, a quicker, less expensive reverse genetics method was needed. Luckily scientists discovered . . .
RNAi

6. The old model: Antisense RNA leads to translational inhibition
mRNA is considered the sense strand
antisense RNA is complementary to the sense strand

7. The old model: Antisense RNA leads to translational inhibition
This can give the same phenotype as a mutant

8. 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

9. genetic interference by double-stranded RNA in
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 sequence-specific silencing and coined the term "RNA Interference".

10. 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)

11. HOW DOES RNAi WORK?
The Central Dogma
DNA (double-stranded)
RNA (single-stranded)
Protein

12. When good RNA goes bad....
Viruses can
make double-
stranded RNA

13. When good RNA goes bad....
Cells sense double-
stranded RNA and
activate a response
called RNAi

14. Understanding how
RNAi works is the
key to using it as a
genetic tool and for
therapy

15. Dicer cuts dsRNA into short RNAs
Vanhecke, D.; Janitz, M. Drug Discov. Today, 2005, 10,
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.

16. 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!

17. How does the vector induce RNAi?
Allows E. coli to produce double-stranded RNA
Requires a specific strain of E. coli

18. 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.

19. E. coli strain HT115(DE3) has a modified lac promoter controlling the transcription of T7 RNA polymerase
T7 RNA polymerase
T7 RNA polymerase

20. When IPTG is added T7 RNA polymerase is expressed -
this transcribes the dsRNA
T7 RNA
polymerase

21. 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