To begin, use a transgenic line expressing GFP and nitro reductase, or NTR, under a rod photoreceptor specific promoter. NTR converts the prodrug metronidazole, or MTZ, into a cytotoxic agent for selective ablation of NTR expressing rods. For GFP monitoring, use a spoon to gently catch one anesthetized tadpole and cautiously place it onto a moist tissue within a small Petri dish.
Observe the GFP fluorescence under a fluorescent stereo microscope and capture photographic images of the eyes. Carefully immerse the Petri dish with the tadpole into a large tank with rearing water until fully awakened. For individual monitoring of retinal degeneration, place each tadpole in a small container containing 30 milliliters of 10 millimolar MTZ, or transgenic siblings in the control solution.
For batch treatment, place the transgenic tadpoles in a larger container with one liter of MTZ, or control solution. Raise the tadpoles for one week at 20 degrees Celsius under darkness to avoid degradation of MTZ. After seven days, observe the GFP under the fluorescent stereo microscope and capture photographic images of the eye.
A decrease in fluorescence intensity, or even a virtual absence of fluorescence indicates the degradation of rods. The eyes of transgenic tadpoles treated with MTZ showed a decrease in GFP fluorescence compared to the controls, which is further confirmed on retinal sections by GFP immuno staining.
