Introduction to C. elegans: Laboratory course

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Introduction to C. elegansLaboratory course

• PRACTICALS

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Mon 25.08.08

• Basic worm handling

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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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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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• 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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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

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Transferring (picking) the worms from NGM agar
plates

• Platinum wire attached to
• a Pasteur pippette
• (glass is melted with flame)
• Eyelash glued to a toothpick
• Chunking pieces of agar

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C. elegans anatomy
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1. RNAi with unc-22 dsRNA
L4

• Place 4 L4 larval stage worms on RNAi plates. You
  can see the effect of unc-22 RNAi in progeny of
  those worms (after 2-3 days).

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2. Dauers

• Transfer
• 3 L4
• CB1370 (daf-2)
• keep one plate at 25 C
• and one plate at 20 C

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3. Mating

• Transfer 3 N2 L4 hermaphrodites and 4 to 6 him-8
  adult males onto a mating plate
• Check the progeny after 2-3 days
• You should have 50 of males in the progeny

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him high incidence of males
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4. Synchronization aka bleaching

• Harvest the worms from contaminated plate into a
  test tube by washing it with 1 ml of M9
• Centrifuge at 2000 gpm for 20 60 sec
• Discard most of the liquid
• Fill the tube to 500 µl with H2O and add bleach
  (prepare the solution in advance!)
• 350 µl of H2O
• 50 µl of 5N NaOH
• 100 µl of 5 NaOCl (or any kind of
  chloride-containing cleaning compound)
• Wait for 5-10 minutes, examine under microscope
  what is going on inside the tube
• Centrifuge at 2000 gpm for 20 60 sec
• Discard most of the liquid, fill the tube with
  sterile M9
• Repeat two last steps 2 times
• Centrifuge at 2000 gpm for 20 60 sec
• Discard most of the liquid, transfer the eggs to
  a new clean NGM plate

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Tue 26.08.08

• assays

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1. Egg laying assay

• L4 larvae were placed on plates with food and
  allowed to mature at 25C overnight to become
  young adults.
• Move the young adults to individual plates that
  either contain or lack food. Allow the worms to
  lay eggs at 25C for 2 hours.
• Count the number of eggs present on the plate.

the egg-laying rate in the presence of abundant
food is significantly higher than in the absence
of food
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2. Levamizole assay

• Transfer 5 young adults onto NGM plates
  supplemented with 10 mM levamisole (tetramisole).
  Observe the behavior of the worms.
• After 5 minutes transfer the animals to a regular
  NGM plate. Wait for one hour, observe the
  behavior.

The cholinergic anthelmintics like levamisole act
on nematode nicotinic AChR located on somatic
muscle cells. It causes hypercontraction of
nematode body-wall muscles, leading to death.
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3. Ethanol assay thrashing

• Take a glass slide and pipet 20 µl M9 onto the
  slide.
• Pick one young adult N2 and put it into the drop.
• Count body bends during one minute
• Repeat the experiment with 0.1 EtOH and 5 EtOH

In liquid, wild type animals exhibit a rhythmic
flexing motion centred on the midpoint of the
body called 'thrashing'. A single thrash is
defined as a complete movement through the
midpoint and back.
Low doses (from to 0.1, 17.4 mM 0.3, 52.3 mM)
elicit hyperactivity, Higher doses (0.5,
87.0 mM to 5.1, 870 mM) decrease motility
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Ethanol

• Ethanol has concentration-dependent effects on
  neural function
• In the low millimolar range, equivalent to the
  drink-driving limit, it has an intoxicating
  action, which is often associated with a
  transient excitability, followed by inhibition

• At concentrations greater than 100 mM, it has
  an anaesthetic action and elicits muscle paralysis

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5. Nile Red

• Nile red stains intracellular lipid droplets red.
  Is also intensely fluorescent, with a strong
  yellow-gold emission when in a lipid-rich
  environment

• Nile Red powder was dissolved in acetone at 500
  mg/ml, diluted in 1X phosphate buffered saline
  (PBS) and added on top of nematode growth media
  (NGM) plates already seeded with OP50 bacteria,
  to a final concentration of 0.05 mg/ml.
• Place worms (N2, daf-2, fat-3) onto these plates
  as eggs or starved L1s (use L1s from bleaching)
  and keep the plates at 25C overnight.
• Their staining phenotypes will be assessed prior
  to starvation at the L4 and the young adult
  stages.
• Fat content will be monitored by fluorescence
  microscopy.

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Wed 27.08.08

• Microscopy slides

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1. Mutants
Observe under the microscope

• bli
• dpy
• lon

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2. Isolation of dauer larvae

• The dauer-specific cuticle and the lack of
  pharyngeal pumping confer resistance to many
  environmental insults, including 1 SDS

• Wash the daf-2 plates (20C and 25 C ) with M9
  to collect the worms into test tubes.
• Centrifuge for 20-60 sec at 2000 rpm and discard
  excess liquid.
• Add 1 ml 1 SDS and wait for 15 minutes.
• Centrifuge for 20-60 sec at 2000 rpm and discard
  excess liquid.
• Pipet the worms onto a new plate and compare the
  amount of survived worms.
• Observe dauer morphology

L4 dauer
Riddle Laboratory UBC Campus Vancouver - Gallery
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2. Making microscopy slides

• (a) Three microscope slides are placed in a row,
  with a 2-3 layers of lab tape on each of the two
  outer slides.
• A drop of 4 agarose in M9 is placed on the
  center slide.
• (b) A fourth microscope slide is rested on the
  lab tape on top of the two outer slides and used
  to flatten the agarose on the center slide.
• (c) The central slide is slid out.
• (d) 5 µl of 20 mM sodium azide in S basal is
  pipetted onto the agarose pad and animals are
  picked into the azide.
• A coverslip is placed atop the animals.

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Embryonal and larval stages
Annie Lam
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Transgenic lines

• 11-7 dat-1GFP

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Transgenic lines

• aex-3GFP - pan-neuronal expression

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Transgenic lines

• myo-2GFP

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Nile red

• Compare the intensity of Nile Red staining in N2,
  daf-2 and fat-3 strains

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Thu 28.08.08

• Conclusions

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• Mating plates
• RNAi worms