The overall goal of this experiment is to deliver a DNA vaccine intradermally into the skin of a laboratory mouse using the DNA Tattooing technique. This is accomplished by the following steps. First, isolate and prepare the plasmid DNA from DH five alpha e coli bacterial culture.
Next, trim and shave the animal's hind leg to expose the skin. Then administer the DNA into the animal's skin by DNA tattooing. Finally, dissect the skin to confirm the antigen expression using confocal microscopy.
This video is produced by the Kong lab at the New York University School of Medicine. With this tattooing technique, plasma DNA encoding and antigen is delivered into the skin cells with an oxalated needle array, where expression of antigen leads to a rapid and robust induction of immune responses. Skin tattooing is a novel intradermal, DNA delivery technique that is safe and cost effective.
The thousands of tiny perforations by the needle may also serve as potent adjuvant. All of this makes the tattooing technique an attractive candidate for administering A DNA vaccine. To begin the experiment, first, prepare the DNA to be delivered intradermally here for better visualization, we will use a eukaryotic plasmid encoding enhanced green fluorescent protein or EGFP.
The plasmid is then transformed into DH five alpha e coli cells and purified using the kyogen and endo free plasmid maxi prep kit. Ideally, the purified plasmid solution should be as concentrated as possible to minimize the volume required for the tattooing process. The DNA solution should be filtered with a low binding 0.22 micron filter and kept under sterile conditions at minus 20 degrees Celsius until use.
Next, we will set up the tattoo equipment. A typical tattoo system consists of a handheld unit, a power supply unit, and a control pedal. In this demonstration, we use a stealth rotary tattoo system due to its compact design and lightweight.
However, any other commercially available rotary tattoo machine should also be adequate. First, connect the handheld unit and the control pedal to the power supply unit according to the manufacturer's instructions. Then set the needle oscillation frequency to approximately 100 hertz.
Next, install the needle into the handheld unit. Note, the tattoo needle should be autoclave and cleaned with 70%ethanol before use. Once the needle is place, attach the plastic grip loosely on the handheld unit.
Use the control pedal to start oscillation of the needle and inspect the needle depth. Adjust the needle depth to an appropriate setting for your experiment. For our mouse experiment, we used a needle depth of one half millimeter.
Finally tighten the Plastic grip and the tattoo machine is ready to be used. Next, we will prepare the animal for skin tattooing. A mixture of ketamine and xylazine is used to anesthetize a bulb sea mouse before shaving.
It is important to check the animal's reflexes to ensure it is properly anesthetized. To shave the animal, we simply use an electrical hair trimmer and a disposable safety razor that are commercially available. First, use the electrical shaver to trim down the hair slowly and carefully.
The residual shorter hair is then removed using the disposable razor. This process should be performed with care to avoid damaging the animal's skin To start the tattooing process, the DNA solution is applied at the tattooing site. The DNA solution can be either directly pipetted onto the skin or loaded into the plastic grip that holds the needle.
In our demonstration, we used a dose of approximately 1.8 micrograms of DNA over an area of one square centimeter. Using a small volume as practical makes the process more manageable. Once the DNA solution is properly applied, place the oscillating needle on the skin and move it gently and slowly.
To avoid unwanted cutting, the needle must be kept perpendicular to the skin surface. Minor bleeding from the capillary vessels is normal. If the bleeding becomes more severe, stop the tattooing and decrease the needle depth.
Continue tattooing for approximately one minute. After the tattoo treatment, the skin at the tattooing site should exhibit inflammation with minimal bleeding. A topical analgesic such as SSD cream or Neosporin ointment should be applied to the tattooing site.
If the animal exhibits signs of discomfort on the following day reapply the analgesic. 48 hours after the tattooing procedure, we checked for EGFP expression in the skin cells. To examine the EGFP signal, the skin of the hind legs was dissected and fixed in 4%Paraform aldehyde at four degrees Celsius overnight.
The whole tissue was examined by confocal microscopy and EGFP Expression in the skin cells was confirmed. This concludes our Demonstration of DNA skin tattooing. We hope you have found this video useful.
Thank you and good luck with your experiments.
