Gene regulation in biological responses Hypothesis testing Differentiation ProliferationApoptosis High throughput 96 well plates array based Single gene.

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?

siRNAs have a defined structure

19 nt duplex

2 nt 3’ overhangs

siRNA

Model for RNAi

RISC

RNAi in mammalian cellssiRNA: short interfering RNAshRNA: short hairpin RNA

shRNA

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

RNAi: High-throughput screen protocol

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

Integrating data from the rat and the mouse for studies of human diseases

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

lentiviral construct for siRNAs

Rubinson et al Nature Genetics, 2003