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Introduction to C. elegans : Laboratory course

Introduction to C. elegans : Laboratory course. University of Tartu Institute of Molecular and Cell Biology Riia 23, Tartu, Estonia 25.08. – 28.08.2008. Organizers and Teaching Staff. LECTURES: Kaja Reisner, University of Tartu & University of Kuopio Suvi Asikainen, University of Kuopio

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Introduction to C. elegans : Laboratory course

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  1. Introduction to C. elegans:Laboratory course University of Tartu Institute of Molecular and Cell Biology Riia 23, Tartu, Estonia 25.08. – 28.08.2008

  2. Organizers and Teaching Staff LECTURES: • Kaja Reisner, University of Tartu & University of Kuopio • Suvi Asikainen, University of Kuopio • Vuokko Aarnio, University of Kuopio • Olli Matikainen, University of Helsinki LABORATORY EXERCISES: • Egon Urgard, University of Tartu • Tanel Jantson, University of Tartu

  3. Course outline LECTURES (Riia 23,lecture room 208 ) Mon 25.08.0810.15 - 11.00 Introduction to C. elegans Tue 26.08.089.15 - 10.00 Genome, mutants and transgenic worms 10.15 - 11.00 Phenotyping - behavioral and pharmacological assays Wed 27.08.08 9.15 - 9.45 C. elegans development 9.50 - 10.20 RNA interference and small RNA molecules 10.25 - 11.00 C. elegans bioinformatics Thu 28.08.089.15 - 9.45 Neurodegenerative diseases 9.50 - 10.20 The ubiquitin-proteasome system 10.25 - 11.00 Biosynthesis and function of fatty acids

  4. Course outline LABORATORY EXERCISES (Riia 23, room 113) Mon 25.08.08 Worm picking: mating plates; RNAi (unc-22); daf-2 for dauers to 25° C Tue 26.08.08 Mutants; thrashing assay; levamisole assay; egg laying assay; synchronization Wed 27.08.08 Slides with: larval and embryonic stages; dauers (1 % SDS test); transgenic GFP-expressing worms, Nile Red worms Thu 28.08.08 Looking at: RNAi worms (twitching), mating plates,pictures taken

  5. Introduction to C. elegans 1st lecture:

  6. Some examples of model organisms • Unicellular – bacteria, yeast, mycoplasma • Multicellular invertebrates – nematodes, insects, plants • Multicellular vertebrates - rodents, fish, birds, nonhuman primates • Morphological oddities – starfish, seasquirts, bats, turtles, flatworms

  7. Most commonly used model organisms Yeast (S. cerevisiae) Worms (C. elegans) Fruitfly (D. melanogaster) Zebrafish (D. rerio) Mustard Weed (A. thaliana) Mouse (M. musculus)

  8. Scorecard C.elegans H.sapiens Chromosomes 5 + 1 22 + 2 Genome Size 97 million 3000 million Encoded Proteins 19,099 ~30,000 Life Span 0.06 years ~80 years Sexes male, herm. male, female Somatic cells 1031, 959 ??? Neurons 381, 302 100,000,000,000 Synaptic connections 5,000 100,000,000,000,000 Body size 1 mm ~170 cm Body weight 5 g ~75 kg Food E.coli Omnivore

  9. it can be handled like a microbe – very amenable togenetic analysis - WHY? • on agar plates • in liquid medium • as frozen stocks • self-fertilization • crosses with males • short life cycle • genome sequence known Dec 11 1998 Vol 282: 5396 C. elegans: Sequence to Biology

  10. easy to observe under microscope- • easy to make mutants- • small size (1 mm) • transparent body • invariant cell number • mutagenesis • DNA microinjection • RNA interference http://130.15.90.245/photos.htm

  11. Nobel Prize in Physiology or Medicine2002 Sydney Brenner John Sulston Robert Horvitz "for their discoveries concerning genetic regulation of organ development and programmed cell death"

  12. “...we must move on to other problems in biology which are new, mysterious, and exciting.” 1962

  13. “...broadly speaking, the fields which we should now enter are development and the nervous system.“ John Sulston

  14. Because • the worm is transparent and • the pattern of differentiation is so rigid • it has been possible to trace the lineage of every single somatic cell in the animal. EUTELY - the pattern of development and cell number is INVARIABLE from worm to worm. 558 cells at hatching, 959 in adult 131 cells in the developing embryo die by APOPTOSIS.

  15. Apoptotic pathways of C. elegans and vertebrates are conserved

  16. Nobel Prize in Physiology or Medicine2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA" Andrew Z. Fire Craig C. Mello

  17. Phylum Nematoda • C. elegans is one of 10,000 known nematodespecies. • The actual number may be more than 100,000. • In terms of individuals, nematodes account for an estimated four of every five animals in the world.

  18. Cl. Secernentea (Phasmidea) _____________________ Cl. Adenophorea (Aphasmidea)

  19. Parasitic nematodes Currently infect about 3 billion people C.elegans is NOT parasitic! Plant parasitic nematodes, such as root knot nematode, cause an estimated 80 billion dollars in crop damage annually. parasites

  20. THE WORM In case of self-fertilization there are ~ 0.1 - 0.3% male worms in the population. http://www.wormatlas.org/handbook/contents.htm

  21. Males Males (5AA;X0) arise from fusion of nullo-X gametes and normal X-bearing gametes. Nullo-X gametes are generated by spontaneous non-disjunction of the X chromosome during meiosis in the germ line.

  22. Nervous system

  23. Worm and neurobiological studies • Depression • Neurodegeneration • Schizophrenia • Insomnia • Addiction • Memory • Learning • etc.

  24. Individual neurons • http://www.wormatlas.org/neurons.htm/neuroappdx.htm http://www.wormatlas.org/neuronsimages/NeuronImageList.jpg

  25. Life cycle ~ 3 days from egg to egg-laying parent

  26. Adverse environmental cues induce dauer arrest in C. elegans normal environment L3 embryo L1 L2 L3 L4 adult dauer • growth arrest • increased fat • extended lifespan adverse environment dauer

  27. daf-2 mutants • have significantly longer lifespan than wild type • form 100% dauer larvae at 25 °C – dauer constitutive

  28. A conserved insulin signaling pathway in C. elegans insulin-like ligands: DAF-28 and INS1-39 PI’s insulin receptor-like P55 AAP-1 PI3K PTEN lipid phosphatase PI3P’s IST-1 IRS-1 kinases PDK-1 on off FOXO transcription factor development metabolism aging & etc.

  29. Maintainance of C .elegans NGM (Nematode Growth Medium) agar plates : • 34.0 g Agar • 2.5 g Peptone • 3.0 g NaCl • Fill up with 1 L H2O • Invert bottle a couple of times • Autoclave the mixture (20 min at 121°C) • Let it cool down to approximately 50°C • Add: • 1 ml Cholesterol/Ethanol (5 g Cholesterol in 1 l 95% Ethanol) • 1 ml 1M MgSO4 • 25 ml 1M KPO4 • 1 ml 1M CaCl2 • Pour NGM plates

  30. OP50 is a uracil auxotroph whose growth is limited on NGM plates. A limited bacterial lawn is desirable because it allows for easier observation and better mating of the worms.

  31. Maintainance of C .elegans • NGM (Nematode Growth Medium) plates • seeded with OP50 Esherichia coli strain • Worm stocks can best be maintained between 15 °C and 25 °C, most typically at 20°C. Worms grow 2.1 times faster at 25°C than at 16°C, and 1.3 times faster at 20 °C than at 16 °C

  32. Long term storage of the worm C. elegans can be stored indefinitely at very low temperature (-70 ~ -100 °C freezer) Freezingsolution: S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl] + 30% glycerin 1:1 with M9 containing worms (preferably starved L1) In the dauer larval stage, it can also be kept at 16 °C for months

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