Genome-wide RNAi screening in Caenorhabditis elegans

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Genome-wide RNAi screening in Caenorhabditis
elegans

• Ravi S. Kamath Julie Ahringer

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What is RNAi?
A cellular mechanism to regulate the expression
of genes, mutant gene products and the
replication of viruses
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(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
  interferencenot 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

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How does this stuff work?The cool movie revisited
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How do you get dsRNA into C.elegans?
Microinjection
Soaking in dsRNA
Feeding bacteria expressing dsRNA
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Advantages of feeding for high-throughput RNAi
screening

• Fast
• Cheap
• Less labor intensive

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Aim of this paper

• provide research community with a rapid
  screening tool
• describe methods for bacterial feeding library
  construction
• Identify new gene functions

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Methods
Cloning Conveniently, Genepairs primers
commercially available - optimized for max.
overlap with coding region - amplify 1000-1500bp
fragments at 5 end of gene
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Construction of feeding library Making the
construct
Need dsRNA to yield effective RNAi phenotypes
Used L4440 (pPD129.36)
MCS
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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

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Construction of feeding library Suitable
Bacterial strain

• Transformed RNAi constructs into HT115(DE3)
• RNase III-deficient strain
• Tetracycline resistant
• Increased transformation efficiency using TSS

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Construction of feeding library
Plate positive clones onto NGM Carb IPTG
plates
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High-throughput phenotype screening
7-10 worms
Clone 3 adult worms to 3 separate wells
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High-throughput phenotype screening Timeline
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High-throughput phenotype screening Analysis of
phenotypes
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Interlude
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Drawbacks

• Some genes hard to target
• Genes whose protein product has a long ½ life
• Nervous system genes difficult to target
• Variability in phenotypes
• Inconsistency between animals
• Phenotypes can resemble hypomorph rather than
  amorph
• Silencing of related genes
• Genes with close homologs can often be abated in
  addition to target

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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

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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

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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

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RNAi screen for novel muscle mutants
Microarray
SAGE
Muscle Expressome

Screen for Disorganized Sarcomeres
Feed Myo-3GFP worms RNAi clones
RNAi Clone Library
Normal myo-3 localization
Abnormal myo-3 localization
Characterize mutants obtained in RNAi screen
Repeat RNAi screen of positive genes to confirm
validity
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