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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. 2001. 1990. 1995. 1998. 2003.

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Genome-wide RNAi screening in Caenorhabditis elegans

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  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 2001 1990 1995 1998 2003 • 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

  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. 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 Advantages of feeding for high-throughput RNAi screening • Fast • Cheap • Less labor intensive

  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. 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? Methods Cloning: Conveniently, Genepairs primers commercially available - optimized for max. overlap with coding region - amplify 1000-1500bp fragments at 5’ end of gene

  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 EcoRVto 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 • 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

  18. How could you speed up this assay? 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

  19. Results of genome-wide screen & library construction • Identified novel gene functions for ~10% of the ~19000 genes screened using N2 worms • Created a functional, rapid means to perform a large-scale RNAi screen • 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 • Found additional loss-of-function phenotypes for 393 genes • In replicates of experiments, found consistent false-positives

  21. RNAi screen for novel muscle mutants Microarray Muscle ‘Expressome’ SAGE + Screen for Disorganized Sarcomeres Feed Myo-3::GFP 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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