We use this protocol to search for novel vaccine candidates against tuberculosis, but I can be applied to other infectious diseases. Compared to mammalian models this method provides a cost-effective procedure for the preliminary screening of novel DNA-based vaccine candidates. Demonstrating the procedure will be Hannaleena Piippo, a technician from our laboratory.
To begin, set the glass capillary into the V-groove in the polar bar and tighten the clamping knob lightly. Move the holder next to the filament and gently push the capillary through the filament and into the polar bar on the other side of the filament. Next, tighten the clamping knobs, set down the safety glass, and press the pull button.
After this, place the needles on a piece of reusable adhesive inside a Petri dish to protect the needle tips. First, prepare the injection mix using 0.5 to 12 micrograms of plasmid per dose. Prepare the appropriate volume of master mix for the number of fish in the group.
Place the capillary needle on the sticky side of a piece of tape on appropriate holder. Next, pipette droplets of plasmid doses onto a piece of laboratory film. After this, use a loading tip to transfer the plasmid drops from the film into the needle.
Pipette slowly and carefully to avoid pipetting air bubbles into the needle. Position the micro manipulator and a light source. Then switch the air pressure tap to the open position.
On the micro injector front panel adjust the pulse length to 10 seconds using timed mode. Then, fine tune the pulse length with the 10-turn period dial. Next, set the needle on to the micro pipette holder of the micro manipulator.
Use tweezers to cut the tip of the needle to allow liquid to be pushed out, and use a microscope to assess the position of the needle. Press the remote foot switch once to verify that a one second pulse pushes a small droplet out of the needle. Then adjust the electroporator settings and connect the tweezers to the electroporator.
To keep the fish in a fixed position during injection use a piece of sponge with a groove cut in the middle as padding. Soak the sponge thoroughly in system water and set it in a Petri dish. Using a plastic spoon, transfer an anesthetized zebrafish to the wet sponge with the fish's ventral side down in the groove.
Under the microscope place the needle at a 45 degree angle close to the zebrafish's dorsal muscle, then find the small spot without scales in front of the dorsal fin and push the needle into the muscle. If any resistance is felt try an adjacent spot. Use the foot switch to gradually inject the plasmid solution into the muscle.
Under the microscope the phenol red should be visible as it enters the muscle tissue. Electroporate the fish immediately after injection by placing the tweezer-type electrodes at each side of the injection site. Press the start button on the electroporator and give it six 40-volt 50 millisecond pulses.
Then, gently transfer the fish to a recovery tank. Finally, after anaethisizing the fish according to the text protocol, use a UV light to see EGFP expression near the injection site. In this protocol, DNA antigens were cloned adjacent to GFP in the expression vector.
Zebrafish were injected with the DNA antigen plasmids intramuscularly with a micro injector, and the injection site was electroporated to improve the intake of plasmids into the cells. For imaging, antigen expression can be visualized with fluorescence microscopy. In this case, expression of all three antigen GFP fusion proteins was detected near the injection site, although their intensity varied.
Five weeks after immunization with mycobacterial antigens the zebrafish were infected with Mycobacterium marinum. QPCR was used to determine the bacterial burden in each fish four weeks after infection. Compared to the control group, the fish immunized with antigen one showed a decreased bacterial loads after infection with M.marinum.
While performing this method it is important to design the plasmid construct carefully, validate expression of the antigen, and practice the injection technique before large-scale experiments. This technique can pave the way for cost effective pre-clinical screening of novel DNA-based vaccine candidates in adult zebrafish. Don't forget that the welfare of the animals is very important, and proper anesthesia is essential while performing this procedure.
