Introduction%20to%20C.%20elegans:%20Laboratory%20course

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Introduction to C. elegansLaboratory course

• University of Tartu
• Institute of Molecular and Cell Biology
• Riia 23, Tartu, Estonia
• 25.08. 28.08.2008

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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

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Course outline
LECTURES (Riia 23, lecture room 208 ) Mon
25.08.08 10.15 - 11.00 Introduction to C.
elegans Tue 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 acids
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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

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Introduction to C. elegans
1st lecture
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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

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Most commonly used model organisms
Yeast (S. cerevisiae)
Worms (C. elegans)
Fruitfly (D. melanogaster)
Zebrafish (D. rerio)
Mustard Weed (A. thaliana)
Mouse (M. musculus)
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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
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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
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• 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
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Nobel Prize in Physiology or Medicine 2002
Sydney Brenner John Sulston Robert Horvitz
"for their discoveries concerning genetic
regulation of organ development and programmed
cell death"
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...we must move on to other problems in
biology which are new, mysterious, and exciting.
1962
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...broadly speaking, the fields which we should
now enter are development and the nervous system.
John Sulston
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• 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.
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Apoptotic pathways of C. elegans and vertebrates
are conserved
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Nobel Prize in Physiology or Medicine 2006
"for their discovery of RNA interference - gene
silencing by double-stranded RNA"
Andrew Z. Fire Craig C. Mello
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Phylum Nematoda

• C. elegans is one of 10,000 known nematode
  species.
• 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.

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Cl. Secernentea (Phasmidea) _____________________
Cl. Adenophorea (Aphasmidea)
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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
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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
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(No Transcript)
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Males

• Males (5AAX0) 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.

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Nervous system
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Worm and neurobiological studies

• Depression
• Neurodegeneration
• Schizophrenia
• Insomnia
• Addiction
• Memory
• Learning
• etc.

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Individual neurons

• http//www.wormatlas.org/neurons.htm/neuroappdx.ht
  m

http//www.wormatlas.org/neuronsimages/NeuronImage
List.jpg
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Life cycle
3 days from egg to egg-laying parent
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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
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daf-2 mutants

• have significantly longer lifespan than wild
  type
• form 100 dauer larvae at 25 C dauer
  constitutive

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A conserved insulin signaling pathway in C.
elegans
insulin-like ligands DAF-28 and INS1-39
PIs
insulin receptor-like
P55
AAP-1
PI3K
PTEN lipid phosphatase
PI3Ps
IST-1
IRS-1
PDK-1
kinases
on
off
FOXO transcription factor
development
aging etc.
metabolism
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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 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

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• 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.

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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 C
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Long term storage of the worm

• C. 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
• 11 with M9 containing worms (preferably starved
  L1)
• In the dauer larval stage, it can
• also be kept at 16 C for months