Zebrafish Morpholino Injection Protocol

Prepared with J. Galloway

2/17/2007

Stock preparation

  1. Morpholinos arrive in a vial at a concentration of 300 nm. Add 75 µl of sterile, distilled water to the vial. Vortex completely. This will yield a 4 mM concentration. Once resuspended, keep morpholinos frozen at -20C.
  2. Prepare two concentrations for injections as described below.
  3. In one, prepare a 0.2mM final concentration by sub-diluting the stock above by 1:20. Add 17 µl of Danieaux’s solution; 1 µl of stock morpholino; 2 µl of phenol red (10x stock). This is a 0.2mM stock in 20 µl.
  4. In the other, prepare a 1mM final concentration by subdiluted the stock morpholino above by 1:4. Add 6.5 µl of Danieaux’s solution; 2.5 µl of stock morpholino; 1 µl of phenol red (10x stock). This is a 1mM stock in 10 µl.
  5. In the future, always thaw morpholinos at 50C.
  6. Note that phenol red is added simply to help see where the injected material resides in the embryo.

Injection protocol

  1. In the Zon lab injection room, turn on the pump that feeds the injectors.
  2. Go to the wall and turn on the register for each injection apparatus (these should be labeled from the wall and correspond to the number on the injector).
  3. Begin to prepare the injector using the following specifications: P-balance: should be around 0 (however, in our experiments, it was -0.2psi). It should be slightly negative so that the injection media does not leak. P-inject: pressure should be between 20-25 psi. This can be toggled, however, to give the right sized injection volume. The injection pressure can vary from 10-30 psi, but first try for around 20. Injection time: should be adjusted to ~300 milliseconds. In order to get the optimal injection volume, adjust the time of injection first.
  4. Cut a pulled pipet tip with sharpened forceps. Go to the highest magnification and cut the tip to a bevel.
  5. Injection volume should be around 1 nl. This translates into an injection “ball” of about 133 µm in diameter. Use a stage micrometer or ocular micrometer to determine this value.
  6. These injection needles have a tiny trench to allow capillary back-filling. Back-fill the pipet by using a special micron-tipped pipet tip add roughly 3-4 µl to the back part of the injection needle.
  7. Ensure there are no bubbles in the injection needle. Inject into (and store in) mineral oil to ensure the needle doesn’t dry out. A single injection of one nanoliter of the 0.2 mM stock is 0.2 mM of morpholino. Can inject twice to get 0.4 mM; 3 times to get 0.6 mM, etc.

Injection protocol

  1. Carefully embed embryos in agar dishes in long, straight lines.
  2. Approach the embryos from a lateral, largely-horizontal orientation. Carefully inject needle into the embryo (note: can inject only into the yolk since it will diffuse into the embryo).
  3. After the desired number of 1 nl pulses, gently remove needle.
  4. Move the plate of embryos rather than the injector. This makes it easier to simply move the injection needle back and forth.

Collecting embryos

  1. The night before embryonic injections, place male and female adults together in mouse cages filled with fresh system water, egg filters, one tree per side. Keep the male and females separated using a plastic partition.
  2. These cages can be stacked on the cart next to the injection room.
  3. The following morning, arrive as close to the morning when the lights come on as possible as they are triggered to spawn by the early morning dawn.
  4. When ready to inject the embryos, remove the plastic partition. Perhaps shine the flashlight over the adults to stimulate them to breed. Eggs should begin falling through the partition.
  5. Collect eggs by changing the fish to a new fresh, outer mouse cage. Take the cage with eggs and pour through a tea filter. Use the squirt bottle with methylene blue to spray the eggs from the tea filter to a new plastic Petri dish. To make the methylene blue solution, simply add a few drops of methylene blue concentrate to system water.
  6. Wait several minutes until the fertilization membrane expands prior to injecting embryos.