Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs

Morpholinos are short, single-stranded redesigned oligonucleotides with differences in their phosphate backbone and sugar structure as compared to natural nucleotides. These features enhance morpholinos’ stability at the physiological pH. Despite these variations, the morpholinos contain the standard nucleobases, which help in designing oligonucleotide complementary to the specific mRNA sequence from a desired target gene to be knocked down.

To perform morpholino-driven gene editing, begin by taking single-celled, cavefish eggs placed inside the wells of an agarose injection plate. Visualize the translucent and round eggs to locate the yolk sac - a fluid-filled structure present inside the egg.

Take a microinjection needle pre-filled with a solution containing designed morpholinos. Inject the solution directly into the egg yolk, ensuring morpholinos enter the cell cytoplasm. Inside the cells, the target gene forms the mRNA, which is transported to the cytoplasm.

Within the cytoplasm, the injected morpholino hybridizes with its complementary target mRNA transcript at the endogenous initiation site and extends beyond it. This process blocks the binding of the larger subunit of the ribosome with the nascent translation initiation complex consisting of small ribosomal subunit complexed with some specific proteins.

Subsequently, the progression of the translation initiation complex is sterically hindered, which stops the translation of mRNA into the protein. Finally, the target protein concentration reduces due to translational blocking by morpholinos.

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.