Gene regulation in biological responses

Hypothesis testing Differentiation ProliferationApoptosis 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 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 cells siRNA: short interfering RNA shRNA: 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 Oct;13(10):

RNAi Microarray Analysis in Cultured Mammalian Cells Spyro Mousses et al., & Kallionemi Olli, Genome Res Oct;13(10):

Proof of principle: Reverse transfection in HEK293 Flp-In cells: EGFP and Effectene 2X 64 spots: EGFP, HsRed, controls Transfection reagent: Effectene 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 Mar;33(3): 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 Activation Qi 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 born Genetically identical rodents may help pinpoint gene function. Science, Vol. 302, Issue 5648, , November 14, 2003

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