BIOMEDICAL PROBLEM
SCREENINGgenome-widesystem-wide
HYPOTHESIS GENERATIONdata analysis
HYPOTHESIS TESTING
high throughput/single gene
Gene regulation in biological responses
Hypothesis testingDifferentiation
Proliferation Apoptosis
High throughput•96 well plates•array based
Single gene•knock-up•knock-down (RNAi)
animal models
“Essentially, you're destroying mRNA in a very targeted manner."
What is RNA interference?
•RNAi is a way to silence gene expression
•To perform RNAi, dsRNA homologous to the targeted gene is made and then introduced into cells
•Any mRNA with high sequence homology to the dsRNA may be silenced
Dicer contains two RNAse III domains
siRNAs
long dsRNA
1) Processing the dsRNA into 21-23 nt fragments
How does RNAi work?
Need to further characterize mammalian RNAi
• How long does it last?
• How much dsRNA is required?
•Which siRNA/shRNA will work?
• Can any region of a gene be effectively targeted?
Delivery, delivery, delivery…...
Practical aspects of RNAi• Biological research
– defining gene function (gene knockout)
• C. elegans and Drosophila genome RNAi projects
– defining biochemical pathways
• microarray screening of RNAi knockouts
• Therapeutic treatment
– cancer
– viral infection
– parasitic infection
Hypothesis testing: high throughput/single gene
Reverse Transfection RNAi + Microarray
High-throughput selection of effective RNAi probes for gene silencing.Kumar R, Conklin DS, Mittal V. Genome Res. 2003 Oct;13(10):2333-40.
RNAi Microarray Analysis in Cultured Mammalian CellsSpyro Mousses et al., & Kallionemi Olli, Genome Res. 2003 Oct;13(10):2341-7.
Proof of principle:Reverse transfection in HEK293 Flp-In cells: EGFP and Effectene
2X 64 spots:
EGFP, HsRed, controls
Transfection reagent: Effectene
Ma rke r 1 Ma rke r 2 Diffe re nceDis ta nce 10 .24 µm 97 .81 µm 87 .57 µm
Inte ns ity C h3-T4 91 25 -66
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0Dis ta n c e (µ m )
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
In te n s ity
P rofile
one spot: ~100 µm
A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.
D A Rubinson, et al & L Van Parijs..
Nat Genet. 2003 Mar;33(3):401-6. Epub 2003 Feb 18.
RNA interference: Functional silencing of genes in transgenic mice derived from lentivirus-injected zygotes.
Generation of Fertile Cloned Rats by Regulating Oocyte ActivationQi Zhou,1,2 Jean-Paul Renard,1* Gaëlle Le Friec,3 Vincent Brochard,1 Nathalie Beaujean,1 Yacine
Cherifi,3 Alexandre Fraichard,3 Jean Cozzi3
2003: First cloned rats bornGenetically identical rodents may help pinpoint gene function.
Science, Vol. 302, Issue 5648, 1179-1179, November 14, 2003
BIOMEDICAL PROBLEM
SCREENINGgenome-widesystem-wide
HYPOTHESIS GENERATIONdata analysis
HYPOTHESIS TESTING
high throughput/single gene
High throughput•96-well•array based
Single gene•knock-up•knock-down (RNAi)
animal models
Summary