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

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

3. (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 1984 1990 1995 19982003 2001

4. How does this stuff work? The cool movie revisited

5. How do you get dsRNA into C.elegans? Microinjection Soaking in dsRNA Feeding bacteria expressing dsRNA

6. Advantages of feeding for high- throughput 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

7. Aim of this paper  provide research community with a rapid screening tool  describe methods for bacterial feeding library construction  Identify new gene functions

8. 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?

9. Construction of feeding library: Making the construct Need dsRNA to yield effective RNAi phenotypes: Used L4440 (pPD129.36) MCS

10. 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

11. Construction of feeding library: Suitable Bacterial strain Transformed RNAi constructs into HT115(DE3) RNase III-deficient strain Tetracycline resistant Increased transformation efficiency using TSS

12. Construction of feeding library Plate positive clones onto NGM + Carb + IPTG plates

13. High-throughput phenotype screening 7-10 worms Clone 3 adult worms to 3 separate wells

14. High-throughput phenotype screening: Timeline

15. High-throughput phenotype screening: Analysis of phenotypes

16. Interlude

17. 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

18. 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?

19. 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 $$$

20. 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

21. RNAi screen for novel muscle mutants + SAGE Microarray Muscle ‘Expressome’ RNAi Clone Library Feed 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