1. BEH. 109: Laboratory Fundamentals in Biological Engineering
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

2. 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

3. The report will be DUE ON MONDAY, APRIL 29th BY 5 PM.
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.

4. RNA Interference - RNAi
BE109 Module 3
Day 2 lecture

5. RNA interference first discovered in Petunias!
Called PTGS, for “Post Transcriptional Gene Silencing”

6. Color changes can be induced by RNAi, or PTGS..
Post transcipt-ional gene silencing

7. Small (21-23 nts) RNA duplexes, with the same sequence as in the silenced gene, were identified as being responsible for knocking down expression

8. So what other organisms can do this thing called PTGS?
“Post Transcriptional Gene Silencing”

9. Arabidopsis thaliana

10. Protozoa can use RNAi for gene silencing
Planaria
Trypanosomes
Hydra

11. RNAi can operate in insects too!

12. Sulston and Horvitz (1977). Develop. Biol. 56, 110-156.

13. Such gene silencing is a natural phenomenon in this organism
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

14. 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!

15. C. Elegans movie

17. C. Elegans grow on agar dishes with E
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!

18. This does not appear to happen in mammalian cells
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)

19. http://www. nature. com/cgi-taf/DynaPage. taf

20. 16,757 have been inactivated by RNAi
19,757 genes
16,757 have been inactivated by RNAi
10% display spontaneous phenotype; this 10% is enriched for conserved genes

21. 16,757 knock down mutants were screened for body fat content
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?

22. C. Elegans movie

23. So…what about RNAi in mammalian cells…

24. May 2001…the first report…

26. RNAi works postranscriptionally……..
How does RNAi work
in mammalian cells?
RNAi works postranscriptionally……..
in key two steps!

27. Model for RNAi
siRNA

28. processing the dsRNA into 21-23 nt fragments
step one:
processing the dsRNA into nt fragments
short-interfering RNA 34 27 21 20 16
Tuschl, 2001

29. Dicer contains two RNAse III domains
long dsRNA
siRNAs

30. siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs

31. step two: the antisense strand of the siRNA guides cleavage
Tuschl, 2002

32. 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

33. cell death Mammals exhibit potent responses to dsRNA P PKR dsRNA
interferon
production
eiF2a P
cell death
apoptosis
Blockage of protein synthesis

34. siRNAs are not recognized
smaller RNAs can escape the PKR pathway
recall that siRNAs are
intermediate effectors
In the RNAi pathway
siRNAs are not recognized
by the PKR!

35. Two ways to get double stranded RNA

37. Rubinson et al Nature Genetics, 2003
lentiviral construct for siRNAs
Rubinson et al Nature Genetics, 2003

39. 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

40. HIV levels can be reduced by 30-50 fold by siRNA!!!

42. What is the killer virus that might sweep the globe?
Will RNAi eventually become an effective and specific antiviral therapy?