Download Genome-wide RNAi screening in Caenorhabditis elegans

Genome-wide RNAi screening
in Caenorhabditis elegans
Ravi S. Kamath & Julie Ahringer
What is RNAi?
A cellular
mechanism to
regulate the
expression of
genes, mutant
gene products
and the
replication of
viruses
(some) History of RNAi
•1984: Stout & Caskey show antisense RNA can be used to silence
gene expression in Mammalian tissue cultures
•1990: Fire & Moerman show antisense RNA can disrupt myofilament
protein encoding genes
•1995: Guo & Kemphues accidentally discover that sense RNA can is
as effective as antisense RNA in gene silencing
•1998: Mello & Fire illustrate that dsRNA is the agent that leads to
potent and specific genetic interference…not ssRNA
•2001: Fraser et al. complete RNAi screen of 90% of chromosome I
•2003: Ahringer & Kamath unveil the results of a genome-wide RNAi
screen
How does this stuff work?
The cool movie revisited
How do you get dsRNA into
C.elegans?
Microinjection
Soaking in dsRNA
Feeding bacteria
expressing dsRNA
Advantages of feeding for highthroughput RNAi screening
•Fast
•Cheap
•Less labor intensive
Disadvantage: Lots of molecular biology work to clone a
fragment of a gene into a feeding vector and then
transform it into an appropriate bacterial strain
Aim of this paper
 provide
research community with a rapid
screening tool
 describe methods for bacterial feeding
library construction
 Identify new gene functions
Methods
Cloning:
Conveniently, Genepairs primers commercially available
- optimized for max. overlap with coding region
- amplify 1000-1500bp fragments at 5’ end of gene
Problem: How do you rapidly get PCR products into
vector for high-throughput analysis? How do you screen
>19,000 genes rapidly and efficiently for RNAi
phenotypes?
Construction of feeding library:
Making the construct
Need dsRNA to yield effective RNAi phenotypes:
Used L4440 (pPD129.36)
MCS
Construction of feeding library:
Making the construct
• Cut L4440 once with
EcoRV and religated
•Cut L4440 with EcoRV to
create blunt ends for 3’
ddTTP addition by TdT
• Recircularized to
eliminate non-tailed
products
• Ligated PCR A-tailed
PCR products directly into
MCS of vector
Construction of feeding library:
Suitable Bacterial strain
Transformed RNAi constructs into HT115(DE3)
•RNase III-deficient strain
•Tetracycline resistant
•Increased transformation efficiency using TSS
Construction of feeding library
Plate positive clones onto
NGM + Carb + IPTG plates
High-throughput phenotype
screening
7-10 worms
Clone 3
adult
worms to 3
separate
wells
High-throughput phenotype
screening: Timeline
High-throughput phenotype
screening: Analysis of phenotypes
Interlude
Drawbacks
Some genes hard to target
1. Genes whose protein product has a long ½ life
2. Nervous system genes difficult to target
Variability in phenotypes
1. Inconsistency between animals
2. Phenotypes can resemble hypomorph rather than amorph
Silencing of related genes
1. Genes with close homologs can often be abated in addition
to target
How rapid is this screen?
• Once the operation is a well-oiled machine you can screen 200
genes/day with 3 people
• Can screen entire genome in 3 months
• Most labor is in manipulating worms & scoring
How could you speed up this assay?
Results of genome-wide
screen & library construction
1. Identified novel gene functions for ~10% of the ~19000
genes screened using N2 worms
2. Created a functional, rapid means to perform a large-scale
RNAi screen
3. Now a mutant analysis tool is available to the whole worm
community….at a cost $$$
Follow-up Screen
•Simmer et al. (2003) used rrf-3 , an
RNAi-hypersensitive strain to re-assay
the RNAi feeding library
1. Found additional
loss-of-function
phenotypes for
393 genes
2. In replicates of
experiments,
found consistent
false-positives
RNAi screen for novel muscle mutants
Muscle ‘Expressome’
Microarray
SAGE
+
Screen for Disorganized
Sarcomeres
Feed Myo-3::GFP worms
RNAi clones
RNAi Clone Library
Normal myo3
localization
Abnormal
myo-3
localization
Repeat RNAi screen of positive
genes to confirm validity
Characterize
mutants obtained in
RNAi screen