The overall goal of this procedure is to deliver immune related genes into skin tissue to delineate the functional role of a specific gene in a cutaneus inflammation. This method can aid in discovering the function of a novel gene in the skin. For the development of new therapies for cutaneus disease such a psoriasis.
The main advantage of this technique is that it provides a cost effective way to express plasmid DNA in mouse skin. To prepare the plasmid DNA inoculate three milliliters of antibiotic supplemented LB medium with the previously transformed and selected bacterial colony. Incubate the culture overnight on a shaker at 37 degrees celsius.
After the overnight incubation dilute the bacterial culture in 250 milliliters of LB medium, supplemented with antibiotics to scale-up the culture. Incubate the culture overnight at 37 degrees Celsius with shaking. Use a high-quality endotoxin free plasmid preparation kit to prepare a large quantity of DNA.
Dilute the obtained plasmid DNA in sterile PBS to a final concentration of one milligram per milliliter. After anesthetizing the mouse, apply veterinary ophthalmologic ointment to its eyes to prevent dryness. Then shave the skin on the back of the animal.
And apply a depilatory cream to dissolve keratin proteins in the remaining hair shafts. Following depilation wipe off the cream with water soaked cotton. Using a sterile cotton swab, soaked in 70%ethanol disinfect the skin surface and mark the target skin area with the previously prepared template Apply 10 microliters of the DNA solution containing 10 micrograms of plasmid DNA to the desired skin area.
To acufect the mouse, hold the acupuncture apparatus perpendicularly to the skin and repetitively puncture the designated surface. Either 100 times over 30 seconds or until the solution applied to the skin absorbs. To achieve successful plasmid DNA expression in the skin tissue, it is important avoid cutting deep into the skin and to make sure the DNA solution is well absorbed.
After completing the procedure place the acufected mouse on a heating pad to maintain its body temperature until recovery from anesthesia. When the mouse regains sufficient consciousness return it to its cage and provide water and food. Closely monitor the mouse for any symptoms of discomfort including behavioral changes, abnormal appearance for the first 48 hours after the procedure.
Mark the flank skin covering the area of two centimeters by two centimeters previously acufected with IL-15deltaE7. Then using a cotton applicator apply Imiquimod cream topically to the designated surface area. To monitor daily changes of the Imiquimod treated dorsal skin, have one researcher record while the second holds the mouse securely and exposes the treated area to the view of the high definition camcorder.
Observe for such changes as silvery scale, plaque or erythema on the treated skin. After euthanizing the mouse as described, make a horizontal incision at the base of the tail using a pair of serological scissors and widen the cut bilaterally towards the base of each hind limb. Then proceed with vertical cuts that align with the flanks and reach each base of the four limbs.
Carefully peel the skin from the underlying issue starting with the tail towards the head of the animal. Using scissors excise the dorsal skin and transfer the obtained sample to a sterile Petri dish. With a scalpel, excise a suitable sample of the target skin.
Then immediately freeze it in liquid nitrogen. To pulverize the skin tissue, place the frozen sample at the center of a previously chilled two compartment tissue pulverizor equipped with handles and a pestle and use a lead hammer on the mortar. Immediately transfer the pulverized tissue into new tubes containing the reagents appropriate for any subsequent analysis.
Begin the staining procedure by heating the slides of the formalin fixed paraffin embedded skin sections prepared as detailed in the text protocol, at 65 degrees celsius for 15 minutes. Immerse the slides twice in xylene for five minutes. Then rehydrate the slides through an ethanol gradient.
Finally, wash the slides in tap water for three minutes before staining. For Immunohistochemical staining, first draw a circle around the tissue with waterproof pen and then incubate the section with blocking solution to exclude non-specific background staining. After blocking, was the side in PBS.
Next pipette the diluted primary antibody onto the section and incubate it for 60 minutes at room temp temperature. Wash the slides thrice in TBST for five minutes. Then overlay one drop of the labeled polymer over the section and incubate the slide at room temperature for 40 minutes.
Afterward, wash the slide thrice in TBST for five minutes changing the solution between washes. Then add 100 microliters of DAB solution onto the tissue sections and allow it to react at room temperature. Until a brown product is observed under the microscope.
Then wash the slides in water to stop peroxidase activity. Counter stain the section in Methyl Green solution, which specifically binds to DNA for five seconds. Wash the slides three times in PBS for five minutes each.
Overlay the stained section with eight microliters of permanent mounting medium and cover it with a cover slip. Finally, image the slides as described in the text protocol. Pictured here is a control mouse and a mouse that was acufected for one hour, two days, five days or seven days prior to when the photograph was taken.
As shown here, the acufection procedure did not cause noticeable skin damage. Furthermore, acufection did not influence either skin thickness or the number of Ki67 positive cells. Further confirming that the procedure does not cause tissue disruption or epidermal cell proliferation.
The expression of CXCL-1 and IL-6 measured using qRT-PCR. Confirms minimal keratinocyte activation observed under acufection. On the other hand, qRT-PCR and ELISA demonstrated the pronounced expression of IL-15 in the skin of mice acufected with the IL-15 gene.
Ultimately, acufection mediated delivery of the IL-15deltaE7 gene to skin tissue prevents the development of flaky skin and neutrophil infiltration Imiquimod treated mice. Once mastered this procedure can be done in 12 minutes if it is performed properly. While attempting this procedure it is important to remember to prepare a new bundle of needles for each acufection ans a batch of high quality plasmid DNA.
Following this procedure, other methods like virus infection can be performed in order to answer additional questions like the function of a novel gene in modulating antiviral immune response. After its development this will pave the way for researchers in the field of immunology to explore the immune response in the skin. After watching this video, you should have a good understanding of how to induce the expression of plasmid DNA in the mouse skin in a cost effective manner.
