Genome-wide RNAi screening in Caenorhabditis elegans Ravi S. Kamath & Julie Ahringer.

Genome-wide RNAi Genome-wide RNAi screening in screening in

Caenorhabditis elegansCaenorhabditis elegans

Ravi S. Kamath & Julie Ahringer

What is RNAi?What is RNAi?

A cellular mechanism to regulate the expression of genes, mutant gene products and the replication of viruses

(some) History of RNAi(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

19841990

19951998

20032001

How does this stuff work?How does this stuff work?The cool movie revisitedThe cool movie revisited

How do you get dsRNA into How do you get dsRNA into C.elegansC.elegans??

Microinjection

Soaking in dsRNA

Feeding bacteria expressing dsRNA

Advantages of feeding for high-Advantages of feeding for high-throughput RNAi screeningthroughput 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 paperAim of this paper

provide research community with a rapid screening tool

describe methods for bacterial feeding library construction

Identify new gene functions

MethodsMethodsCloning:

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: Construction of feeding library: Making the constructMaking the construct

Need dsRNA to yield effective RNAi phenotypes: Used L4440 (pPD129.36)

MCS

Construction of feeding library: Construction of feeding library: Making the constructMaking 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: Construction of feeding library: Suitable Bacterial strainSuitable Bacterial strain

Transformed RNAi constructs into HT115(DE3)

•RNase III-deficient strain

•Tetracycline resistant

•Increased transformation efficiency using TSS

Construction of feeding libraryConstruction of feeding library

Plate positive clones onto NGM + Carb + IPTG plates

High-throughput phenotype High-throughput phenotype screeningscreening

7-10 worms

Clone 3 adult worms to 3 separate wells

High-throughput phenotype High-throughput phenotype screening: Timelinescreening: Timeline

High-throughput phenotype High-throughput phenotype screening: Analysis of phenotypesscreening: Analysis of phenotypes

InterludeInterlude

DrawbacksDrawbacksSome 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?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 Results of genome-wide screen & library constructionscreen & 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 ScreenFollow-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

+

SAGE Microarray Muscle ‘Expressome’

RNAi Clone LibraryFeed Myo-3::GFP worms

RNAi clones

Normal myo-3 localization

Abnormal myo-3 localization

Screen for Disorganized

Sarcomeres

Repeat RNAi screen of positive genes to confirm validity

Characterize mutants obtained in RNAi screen