Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs
Transcrição
On the day of the injection, use a glass pipette to transfer single-celled eggs into the injection plate, filling up to five rows of the injection plate with 30 to 40 eggs per row. Keep the eggs hydrated with a small amount of fish system water. Thaw morpholino on ice, and prepare the injection solution so that 400 picograms of morpholino is injected per egg, by using a long Microloader pipette tip or adding a 2 to 4-microliter bolus to the end.
When all of the needles have been loaded, use forceps to trim the excess length from the injection needle tips, and mount the first needle into a micromanipulator connected to a picoliter microinjector. Then, place the injection dish under a dissecting microscope and use the micromanipulator to penetrate each egg with the needle, before injecting 1 nanoliter of morpholino injection solution directly into the yolk.
