To begin, take a sharpened capillary, use a micro loader tip to load 2 to 10 microliters of the CRISPR Cas9 ribonucleoprotein complex or control solution. Afterward, place the filled capillary into the micro injector handler. Break off the capillary tip with fine forceps to adjust the ejection volume at 10 nanoliters.
Position one cell stage embryos on a grid glued in a 100 millimeter Petri dish with 3%polysucrose solution. Using the micro injector, and under a stereo microscope, inject 10 nanoliter of the solution into the cortical region, specifically at the animal pole level under the cytoplasmic membrane. After 24 hours, visualize fluorescein, lysine, dextrin under a fluorescent stereo microscope, and select well-injected rho crispant embryos.
Caspase 3 labeling of rho crispant tadpole retinas showed that some rods undergo apoptosis. Histological staining revealed global preservation of nuclear layers, but a severe shortening of photoreceptor outer segments. Further immuno staining analysis with photoreceptor markers showed the degeneration of rod outer segments.
