Download Gene regulation in biological responses

Gene regulation in biological responses
BIOMEDICAL
PROBLEM
HYPOTHESIS
TESTING
high throughput/
single gene
SCREENING
genome-wide
system-wide
HYPOTHESIS
GENERATION
data analysis
Hypothesis testing
Differentiation
High throughput
•96 well plates
•array based
Single gene
•knock-up
•knock-down
(RNAi)
animal models
Proliferation
Apoptosis
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
“Essentially, you're destroying mRNA in a very targeted manner."
How does RNAi work?
1) Processing the dsRNA into 21-23 nt fragments
long dsRNA
siRNAs
Dicer contains two RNAse
III domains
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
Model for RNAi
siRNA
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. 2003 Oct;13(10):2333-40.
RNAi Microarray Analysis in Cultured Mammalian Cells
Spyro 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
one spot: ~100 µm
Marker 1
10.24 µm
91
Distance
Intensity Ch3-T4
Marker 2
97.81 µm
25
Difference
87.57 µm
-66
Profile
Inte ns ity
250
200
150
100
2X 64 spots:
EGFP, HsRed, controls
Transfection reagent: Effectene
50
0
0
10
20
30
40
50
60
Dis ta nc e (µm)
70
80
90
100
Integrating data from the rat and the mouse for studies of human diseases
RNA interference: Functional silencing of genes in transgenic
mice derived from lentivirus-injected zygotes.
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):4016. Epub 2003 Feb 18.
2003: First cloned rats born
Genetically identical rodents may help pinpoint gene function.
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
Science, Vol. 302, Issue 5648, 1179-1179, November 14, 2003
Summary
High throughput
•96-well
•array based
Single gene
•knock-up
•knock-down
(RNAi)
animal models
BIOMEDICAL
PROBLEM
HYPOTHESIS
TESTING
high throughput/
single gene
SCREENING
genome-wide
system-wide
HYPOTHESIS
GENERATION
data analysis
lentiviral construct for siRNAs
Rubinson et al Nature Genetics, 2003