Acufection-Mediated Delivery of Immune-Related Genes into Mouse Skin Tissues

Overview

The video demonstrates a method for directly transferring the interleukin-15 encoding plasmid DNA into the skin tissue of an interleukin-15 deficient mouse model through acupuncture using microneedles. These microneedles generate micropunctures in the outermost skin layer, facilitating the absorption and transfection of plasmid DNA into the epidermal cells, including keratinocytes, within the skin tissue.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. 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 nonpharmaceutical 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.

2. 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).

Representative Results

Figure 1. Illustration of Acupuncture Needles. (A) The acupuncture needle (36 G x 0.5") is composed of a handle and a needle end. (B) Ten acupuncture needles are bound in a bundle using adhesive tapes. The length of the bundle is approximately 4 cm as measured by the ruler (lower panel).

Materials

Name	Company	Catalog Number	Comments
Accu Handy Needle	Accu	36Gx0.5	Medical device
NairTM Lotion	Church & Dwight	N/A	Use to remove hair
Shandon Xyline substitute	Thermo Fisher Scientific	6764506	Deparaffinization
Avertin (2,2,2-Tribromethanol)	Sigma-Aldrich	T4,840-2	Anesthesia
2-methyl-2-butanol	Sigma-Aldrich	152463	Solvent for the dissolution of avertin
QIAGEN® Plasmid Midi Kit	QIAGEN	12143	Purification of plasmid DNA from E. coli
Mouse IL-15/IL-15R Complex ELISA Ready-SET-Go kit	eBioscience	88-7215	Measure IL-15 protein