intorduction to c elegans laboratory course

Introduction to C. elegans:Laboratory courseUniversity of TartuInstitute of Molecular and Cell BiologyRiia 23, Tartu, Estonia25.08. 28.08.2008Organizers and Teaching StaffLECTURES: Kaja Reisner, University of Tartu & University of Kuopio Suvi Asikainen, University of Kuopio Vuokko Aarnio, University of Kuopio Olli Matikainen, University of HelsinkiLABORATORY EXERCISES: Egon Urgard, University of Tartu Tanel Jantson, University of TartuCourse outlineLECTURES (Riia 23, lecture room 208 )Mon 25.08.08 10.15 - 11.00 Introduction to C. elegansTue 26.08.08 9.15 - 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 28.08.08 9.15 - 9.45 Neurodegenerative diseases 9.50 - 10.20 The ubiquitin-proteasome system 10.25 - 11.00 Biosynthesis and function of fatty acidsCourse outlineLABORATORY EXERCISES (Riia 23, room 113)Mon 25.08.08 Worm picking: mating plates; RNAi (unc-22); daf-2 for dauers to 25 CTue 26.08.08 Mutants; thrashing assay; levamisole assay; egg laying assay; synchronizationWed 27.08.08 Slides with: larval and embryonic stages; dauers (1 % SDS test); transgenic GFP-expressing worms, Nile Red wormsThu 28.08.08 Looking at: RNAi worms (twitching), mating plates,pictures takenIntroduction to C. elegans1st lecture: 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 Most commonly used model organismsYeast (S. cerevisiae)Worms (C. elegans)Fruitfly (D. melanogaster)Zebrafish (D. rerio)Mustard Weed (A. thaliana)Mouse (M. musculus)ScorecardWHY?it can be handled like a microbe very amenable to genetic analysis - on agar plates in liquid medium as frozen stocks self-fertilization crosses with males short life cycle genome sequence knownDec 11 1998 Vol 282: 5396 C. elegans: Sequence to Biology easy to observe under microscope- easy to make mutants- small size (1 mm) transparent body invariant cell number mutagenesis DNA microinjection RNA interferencehttp:/130.15.90.245/photos.htmfor their discoveries concerning genetic regulation of organ development and programmed cell death.broadly speaking, the fields which we should now enter are development and the nervous system.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. 558 cells at hatching, 959 in adult131 cells in the developing embryo die by APOPTOSIS.Apoptotic pathways of C. elegans and vertebrates are conservedfor their discovery of RNA interference - gene silencing by double-stranded RNAAndrew Z. FireCraig C. MelloPhylum NematodaCl. Secernentea (Phasmidea) _Cl. Adenophorea (Aphasmidea)Parasitic nematodesCurrently infect about 3 billion people Plant parasitic nematodes, such as root knot nematode, cause an estimated 80 billion dollars in crop damage annually.THE WORMIn case of self-fertilization there are 0.1 - 0.3% male worms in the population.http:/www.wormatlas.org/handbook/contents.htmMalesMales (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.Nervous systemWorm and neurobiological studies Depression Neurodegeneration Schizophrenia Insomnia Addiction Memory Learning etc.Individual neuronshttp:/www.wormatlas.org/neurons.htm/neuroappdx.htmhttp:/www.wormatlas.org/neuronsimages/NeuronImageList.jpgembryoL1L2L3L4adultnormal environment growth arrest increased fat extended lifespandaueradverse environmentL3dauer Adverse environmental cues induce dauer arrest in C. elegans daf-2 mutants have significantly longer lifespan than wild type form 100% dauer larvae at 25 C dauer constitutivedevelopmentaging & etc.metabolisminsulin-like ligands:DAF-28 and INS1-39IST-1AAP-1PDK-1kinasesinsulinreceptor-likePTEN lipidphosphataseFOXOtranscriptionfactorIRS-1P55PI3KPIsPI3PsonoffA conserved insulin signaling pathway in C. elegansNGM (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 121C) Let it cool down to approximately 50C 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 Maintainance of C .elegans 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. 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 20C. Worms grow 2.1 times faster at 25C than at 16C, and 1.3 times faster at 20 C than at 16 CLong term storage of the wormC. elegans can be stored indefinitely at very low temperature (-70 -100 C freezer)Freezing solution: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 canalso be kept at 16 C for months