genome-wide rnai screening in caenorhabditis elegans ravi s. kamath & julie ahringer
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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 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: Analysis of phenotypesscreening: Analysis of phenotypes
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