Development of an Isolated, in Vitro C. elegans Gonad Preparation

1
Development of an Isolated, in Vitro C. elegans
Gonad Preparation

• Adam Broslat
• Advisor Dr. Kevin Strange
• Professor of Anesthesiology and Pharmacology

2
What are C. elegans ?

• 1 mm
• dwells in the soil
• completely sequenced genome
• simple body plan

3
Design Project

• Goal
• design an isolated, in vitro C. elegans gonad
  preparation protocol.

4
Purposes

• for the purpose of characterizing the molecular
  mechanisms of heterologous cell-to-cell
  communication using quantitative microscopy
• This includes Voltage sensitive dyes, pH
  sensitive dyes, Ca sensitive dyes, etc.

5
1st Phase Micro-dissection procedure

• The nematode's gonad will be isolated in such a
  way not to harm the physiology of the gonad.
  Gonad operates independently of the worm
• Problems
• The intestines cloud the view of gonad
• after dissection and gonad does not completely
• remove itself without manipulation.

6
Solutions to Date

• Incisions made with a modified injection needle
  (red line)
• Gonad is half extracted through depressurization
• The other half is forced by suction using
  micropipettes

7
2nd Phase Functional Buffer

• The worm and/or gonad must be placed in a buffer
  solution that promotes normal gonad function
• Problems
• Worms are extremely active in buffer.
• Buffer allows floating and movement.

8
Buffer Recipes
9
Worm Response to Buffer

• Any of the previous listed buffers would sustain
  function to a point.
• The worm gonad needs the the salts to mimic the
  interstitial fluid of the worm.
• The gonad must also have an energy source . .
  .this is where the glucose comes in.

10
Solutions to Date

• .1 Tricaine and .01 Tetramasole anesthetic was
  added to chilled buffer for stabilization.
• Veterinary glue was used on the glass of
  perfusion chamber to secure gonad.

11
3rd Phase - Imaging

• optimal dye loading on perfusion chamber
• DIC image acquisition
• Imaging with argon laser confocal microscope
• Tie the process together and formalize protocol