Development of an Economical DNA Delivery System by “Acufection” and its Application to Skin Research

Female mice at 8 – 12 weeks of age were used for this study. C57/BL6 wild-type (WT) and IL-15 deficient (Il15^-/-) mice were purchased from National Laboratory Animal Center (NLAC), Taiwan and Taconic Farm, respectively. IL-15-deficient (Il15-/-) andIL-15-dominant (Il15+/- and Il15+/+) showed a 1:3 ratio in the second generation from the cross of Il15+/- heterozygotes. The genotype of Il15-/- mice was confirmed by PCR analysis. Mice were maintained in the Specific Pathogen Free (SPF) facility at Laboratory Animal Center (LAC), National Taiwan University (NTU) College of Medicine. All animal procedures including the anesthetic materials and methods were performed in accordance with the animal protocol approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC) (Affidavit of Approval of Animal Protocol 20130225).

1. Preparation of Acupuncture Needles

NOTE: Acupuncture needles are medical devices composed of a handle and a needle end (Figure 1A). The diameters of the acupuncture needle ranged from 0.2 mm (36 G) to 0.35 mm (28 G). We select the finest point of the needle to avoid potential excessive cell damage during pricking the skin. There are 4 different lengths of the 0.2-mm needle including 13 mm (0.5 in), 25 mm (1.0 in), 40 mm (1.5 in) and 50 mm (2.0 in) in length. A bundle of 10 needles with 13 mm length provided the best comfortable grip during oscillating the needle on C57BL/6 mouse skin. The parameters could be modified if the method is applied to the skin of larger animals or performed by researchers with larger hands.

1. Remove acupuncture needles from the sterile, non-pyrogenic package. Place the needles on a sterile drape.
2. Use adhesive labeling tape to bind 10 needles together in a bundle (Figure 1B).
   NOTE: The 10 needles are geometrically arranged to form a cylinder shape. For convenience, use a piece of paraffin film to stabilize the arrangement before applying the adhesive tape. Make sure all the needle points are on the same plane. Using a bundle instead of a single needle facilitates maximal DNA infusion by increasing the contact area between the needles and the skin.

2. Preparation of a Large Amount and Endotoxin-free Plasmid DNA

1. Transform chemically competent bacterial cells with the plasmid DNA.
2. Select and inoculate a single bacterial colony in 3 mL of LB medium containing antibiotics and incubate at 37 °C in a shaker overnight.
3. Scale up the culture by diluting the bacterial culture at 1:100 in 250 mL of LB medium with antibiotics and shaking overnight at 37 °C.
4. Prepare a large quantity of DNA by using a high quality, endotoxin free plasmid preparation kit following the manufacturer's instructions.
   NOTE: Acufection protocol requires high purity and a large quantity of plasmid DNA. Use of a high quality, endotoxin free and column-purified plasmid DNA is recommended.
5. Elute DNA in sterile water. Store DNA in small aliquots at -20 °C until ready for acufection.
   NOTE: Avoid repeated freeze-and-thaw of DNA to ensure consistent plasmid DNA expression.
6. Dilute plasmid DNA to 1 mg/mL in sterile PBS for acufection.

3. Procedure for Acufection

NOTE: The optimal amount of DNA and the area of the target skin surface may vary for different genes and need to be further optimized. The pricking force and number of times to loosen the horny layer of the skin also may vary depending on the skin thickness. These conditions must be carefully determined after evaluating the expression level of acufected gene transcripts by qRT-PCR.

1. Weigh the mice and administer tribromoethanol (400 μg per gm body weight) by peritoneal injection.
   NOTE: 2,2,2-tribromoethanol is an injectable anesthetic agent that was commonly used in mice. Solutions are made by dissolving a non-pharmaceutical grade 2,2,2-tribromoethanol (2.5 g) in distilled water (200 mL) containing 2.5% of 2-methyl-2-butanol.
2. Use vet ophthalmologic ointment on eyes to prevent dryness under anesthesia.
   NOTE: Anesthetic effect will be induced in 1 – 2 min. Check the toe pinch reflex to ensure sufficient depth of anesthesia before operation.
3. Shave the dorsal flank skin and apply depilatory cream to dissolve keratin proteins in the hair shaft.
   NOTE: Use of depilatory cream to remove hair will facilitate infusion of DNA into the skin.
4. Use water soaked cotton balls to wipe off the depilatory cream.
   NOTE: The depilatory cream is soluble in water. Complete removal of the cream will prevent a greasy surface and ensure a better pricking result.
5. Use a sterile cotton swab soaked in 70% ethanol to disinfect the skin surface.
6. Mark the target area (1 cm x 1 cm) on the depilated skin with a pre-measured stencil. NOTE: Marking the target site will help to apply the needle bundle up-and-down within a defined area. This is important to ensure delivery of the same amount of DNA to a specific surface area to minimize experiment-to-experiment variation.
7. Place a 10 μL drop of plasmid DNA (10 μg) onto the marked skin.
   NOTE: The amount of DNA for acufection varies with different genes and the type of expression vector. The amount of DNA used should be carefully titrated before conducting the experiment of interest. The 10 μL is a small liquid drop and it sits well on the shaved and depilated skin without rolling down while pricking. Include a control group of mice treated with 10 μL empty vector DNA to compare with mice acufected with the study gene.
8. Hold the acupuncture needle bundle (Figure 1B) and prick the marked surface with an up-and-down motion for 100 times or until the moisture from DNA in PBS on the skin disappears. NOTE: Some redness of the skin surface may occur. Avoid making deep cuts that bleed. The number of up-and-down pricking motions on the skin surface is operably determined when the moisture from DNA in PBS (10 μL) disappears on the skin. We decided 100 times in 30 s because it has given the most consistent results from acufection-delivered genes. If applicable, the needles can be reused for up to 6 target skin pricks (1 cm x 1 cm each). Rinse the needles in 70% ethanol and PBS between acufection. Change to new needles when they are dull or for different plasmid DNA to avoid cross contamination.
9. Place acufected mice on a heating pad to maintain the body temperature until awake.
10. Return mice to their cage when they have regained sufficient consciousness. Note: Keep acufected mice in separate cage (less than 5 animals per cage) from the cage of un-acufected mice.
11. Put water bottle in place and return the cage to the IVC (individually ventilated cage) rack.

4. Post-operative Care

1. For the first 48 h after the procedure, closely monitor mice for any discomfort including behavioral changes (restlessness, agitation or eating disorder) or abnormal appearance (rough hair coat or hunched posture).

5. Imiquimod (IMQ) Treatment of IL-15ΔE7-acufected Skin

NOTE: Transcriptional expression and protein production of acufected gene are detectable on day 3. Mice acufected with plasmid of interest can be treated with different types of stimulants 3 days after transfection. We illustrate here by treating the IL-15ΔE7-acufected mouse skin with IMQ cream. IMQ is an imidazoquinolin amine approved for treating external genital and perinatal warts¹³. Topical IMQ treatment is shown to induce human psoriasis-like skin disorders in mice with the manifestation of flaky skin, epidermal proliferation and dermal neutrophil infiltration^12,14,15 .

NOTE: The plasmid vector contained full-length mouse IL-15 cDNA, under the regulation of elongation factor-1 α (pEF), flanked with an IL-2 signal peptide and a FLAG tag at the C-terminus (pEF-IL-15, 5859 base pairs), which was constructed as described by Bamford et al.¹⁶. The plasmid is used as an IL-15 template to delete the first 16 amino acids at residues 33 – 48 in exon 7 of the IL-15 gene for IL-15ΔE7 (pEF-IL-15ΔE7, 5,811 base pairs) by SOE (synthesis by overlap extension) PCR method¹⁷. Bacterial colonies that were successfully transformed with IL-15ΔE7 were verified by restriction enzyme digestion and followed by DNA sequencing^12,18. A large quantity of endotoxin-free plasmid DNA was prepared as described in the protocol step 2.

1. Anesthetize acufected mice by intraperitoneal injection of tribromoethanol (400 μg per gm body weight) and apply vet ophthalmologic ointment on eyes to prevent dryness under anesthesia. Check the toe pinch reflex to assure sufficient depth of anesthesia before the start of the operative procedures. NOTE: Since a 2 cm x 2 cm skin will be treated for IMQ, 2 doses of pIL-15ΔE7 plasmid DNA (10 μg in 10 μL PBS per dose) is acufected on 2 target sites (1 cm x 1 cm each).
2. Mark flank skin (2 cm x 2 cm) covering the acufected area.
3. Use Q-tip applicator to topically apply IMQ cream on the marked surface area. Note: step 5.1 is only carried out for the first dose (60 mg/dose). The next consecutive doses are applied to non-anesthetized mice.
4. Document the changes of IMQ-treated dorsal skin daily with a high definition camcorder. Note: One person holds the mouse while another is recording. Still pictures are shot and edited at a later time.
5. Euthanize mice by injecting an overdose of tribromoethanol (800 μg per gm body weight) followed by cervical dislocation on day 4 or day 7 after IMQ treatment. Skin is excised and processed (Steps 4.1 – 4.5).

6. Homogenate of Mouse Skin

1. Use a pair of surgical scissors to make a horizontal cut from the base of the tail. Proceed bilaterally to the base of the hindlimb and continue to cut vertically along the flank to the base of the forelimb.
2. Carefully peel the skin from the underlying tissue from posterior to the anterior. Use the scissors to cut off the dorsal skin. Place the skin on a sterile Petri dish.
3. Use a scalpel to excise target skin and freeze it in liquid nitrogen immediately. Note: The same size of acufected skin without (1 cm x 1 cm) or with IMQ treatment (2 cm x 2 cm) is fixed for each mouse to minimize experiment-to-experiment variations.
4. Place the frozen skin tissue at the center of a pre-chilled 2-compartment mortar with handles and a pestle. Use a lead hammer on the mortar to pulverize the tissue.
5. Quickly place the pulverized tissue to a new tube containing 500 μL of cell lysis reagent for RNA extraction or to a tube containing 50 μL of PBS and 1x protease inhibitor cocktail to obtain protein lysate.

7. H&E and Immunohistochemical Staining of Skin Section

1. Place the skin tissue in a cassette and immerse it in 4% paraformaldehyde overnight.
2. Embed and section tissue, which in our case was performed by the Pathology Laboratory at the LAC, NTU. Note: The section is cut at a thickness of 5 μm. Place tissue sections onto positively charged slides.
3. Heat the slides at 65 °C for 15 min.
4. Place the slides in a staining rack. Immerse the rack into corresponding tank containing xylene substitute for 5 min twice followed by sequential immersion in 100%, 95%, 80%, 75%, 60% and 50% ethanol (5 min each) for rehydration. Immerse the slides in tap water for 3 min before staining.
5. Perform hematoxylin and eosin staining. Note: H & E stain was performed upon request at Pathology Laboratory at the LAC, NTU.
6. For immunohistochemical stain, block the section with block solution at room temperature for 5 min to reduce non-specific background staining. Note: The block solution is commercially developed with immunostaining techniques. No animal serum is contained in this product.
7. Dip the slide in PBS to rinse out the block solution.
8. Dilute the block solution in PBS at 1:10 and add 2% fetal bovine serum (FBS) (diluent solution).
9. Pipette the appropriately diluted primary antibody onto the section and incubate for 60 min at room temperature. Note: Antibodies are diluted in diluent solution. Antibody concentrations for this staining have been optimized at 1:800 and 1:200 for Ki67 (clone SolA15) and Ly6G (clone 1A8), respectively. These concentrations are found to show distinct stain without background staining.
   1. Stain one serial section in the same run with the same solution omitting the primary antibody to control for non-specific binding of the secondary antibody.
10. Wash slides three times in TBS plus 0.1% Tween-20 (TBST solution) for 5 min each. Change solution between washes.
11. Pipette one drop of labeled polymers to each section and incubate at room temperature for 40 min. Note: The labeled polymer is commercially prepared by combining amino acid polymers with peroxidase and secondary antibody which is reduced to a Fab' fragment. The reagent is ready to use for immunohistochemical staining of mouse tissue sections.
12. Wash slides three times in TBST for 5 min each. Change solution between washes.
13. Pipette 3,3'-diaminobenzindine tetrahydrochloride (DAB) solution onto tissue section and let stand at room temperature until a brown precipitate is visible under a bright field microscopy. Note: The time for DAB to precipitate in the presence of peroxidase is around 30 min.
14. Immerse slides in water to stop peroxidase activity. Dip slides in methyl green solution for nuclear staining. Wash slides three times in PBS for 5 min each.
15. Place a coverslip over the stained section that is covered with permanent mounting medium (8 μL per section).

8. Photograph and Analysis of Stained Tissue Section

1. Visualize H &E stained and IHC tissue sections under a bright field microscope. Capture images using charge coupled device (CCD) camera attached to the microscope. For quantitative analysis, imaging software can be used
   1. Scan the whole stained sections using a scanscope with a 20X objective.
   2. Measure the epidermal thickness within a 1-mm segment of the epidermis and count the number of Ki67-immunoreactive cells within the segment.
      NOTE: Quantitative analysis is performed using microscopy automation & image analysis software. Epidermal thickness is defined by the distance between the basal layer and the outermost layer of the epidermis.
   3. Count the number of Ly6G-immunoreactive cells in the dermal area (230 x 270 μm² rectangle) immediately below topical IMQ-treated skin.