Biol 568Advanced Topics in Molecular Genetics

3/16/05

Click here to start

Table of Contents

Biol 568Advanced Topics in Molecular Genetics

Gene silencing by siRNA

Small Interference RNAs

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

RNAi: two step process

RNAi: two step process

RNAi: two step process

Small regulatory RNAs

Micro RNAs: C. elegans

Are all small RNAs processed through the same pathway?

Dicer,the common link

RNAi & viral infection

Viral inhibitors essential for pathogenesis

RNAi as an antiviral therapy

RNAi as an antiviral therapy

Introducing RNAi in mamalian cells

RNAi as an antiviral therapy

RNAi silencing of CD4 to prevent HIV entry and subsequent replications

Small Interference RNAs

The transcriptome

The ìnonspecific repeatsî

The ìnonspecific repeatsî

Heterochromatin

S. pombe mutants

shRNA and heterochromatin

shRNA and heterochromatin

shRNA and heterochromatin

shRNAs regulate heterochromatin formation

Small Interference RNAs

Proof of concept

Cotransfection Assays:

Figure : Suppression of Hyg/EGFP

Suppression of Hyg/EGFP

Ability to repress expression of the reporter gene

Effect of Si RNA is specific

siRNA effect is dose dependent

Presence of siRNA

Efficiency of siRNA

Efficiency of siRNA

Interference of siRNA effect by U-overhangs

Multiple siRNA cassettes

Silencing of endogenous genes

SiRNA EBNA-1/Ori-p system

Silencing of endogenous genes

siRNA advantages:

Viral-mediated silencing of targets

siRNA construct

Fig 1A. Plasmid construct

Cotransfection analyses

Reduced EGFP is due to presence of siGFP RNA

Reduced EGFP is due to specific effect of siGFP

GFP silencing is siRNA specific

Other siRNA constructs

Can virally expressed siRNA reduce expression of endogenous targets?

Effect of siRNA on endogenous genes

Effect of siRNA on endogenous genes

Author: Alan H. Christensen

Home Page: http://mason.gmu.edu/~achriste/568Webpage.html

Download presentation source