1 / 22

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.

afi
Télécharger la présentation

Genome-wide RNAi screening in Caenorhabditis elegans

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related