This work was supported by grant from the Ministry of Science and Technology (MOST 103-2633-B-002-002; 104-2320-B-002-048). We thank Drs. Betty Wu-Hsieh and Chien-Kuo Lee at NTU, Leigh Zerboni at Stanford University and Dr. Peter Hoffmann at the University of Hawaii for reading the manuscript, Yun Chien at NTU for technical assistance, and Dr. Wen-Chi Wei at Agriculture Biotechnology Research Center at Academia Sinica for technical advice.

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.
<ol>
	<li>Remove acupuncture needles from the sterile, non-pyrogenic package.&#160;Place the needles on a sterile drape.</li>
	<li>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.</li>
</ol>
2. Preparation of a Large Amount and Endotoxin-free Plasmid DNA
<ol>
	<li>Transform chemically competent bacterial cells with the plasmid DNA.</li>
	<li>Select and inoculate a single bacterial colony in 3 mL of LB medium containing antibiotics and incubate at 37 &#176;C in a shaker overnight.</li>
	<li>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 &#176;C.</li>
	<li>Prepare a large quantity of DNA by using a high quality, endotoxin free plasmid preparation kit following the manufacturer&#39;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.</li>
	<li>Elute DNA in sterile water. Store DNA in small aliquots at -20 &#176;C until ready for acufection. 
	NOTE: Avoid repeated freeze-and-thaw of DNA to ensure consistent plasmid DNA expression.</li>
	<li>Dilute plasmid DNA to 1 mg/mL in sterile PBS for acufection.</li>
</ol>
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.
<ol>
	<li>Weigh the mice and administer tribromoethanol (400 &#956;g 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use a sterile cotton swab soaked in 70% ethanol to disinfect the skin surface.</li>
	<li>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.</li>
	<li>Place a 10 &#956;L drop of plasmid DNA (10 &#956;g) 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 &#956;L 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 &#956;L empty vector DNA to compare with mice acufected with the study gene.</li>
	<li>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 &#956;L) 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.</li>
	<li>Place acufected mice on a heating pad to maintain the body temperature until awake.</li>
	<li>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.</li>
	<li>Put water bottle in place and return the cage to the IVC (individually ventilated cage) rack.</li>
</ol>
4. Post-operative Care
<ol>
	<li>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).</li>
</ol>
5. Imiquimod (IMQ) Treatment of IL-15&#916;E7-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-15&#916;E7-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 infiltration12,14,15 .
NOTE: The plasmid vector contained full-length mouse IL-15 cDNA, under the regulation of elongation factor-1 &#945; (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-15&#916;E7 (pEF-IL-15&#916;E7, 5,811 base pairs) by SOE (synthesis by overlap extension) PCR method17. Bacterial colonies that were successfully transformed with IL-15&#916;E7 were verified by restriction enzyme digestion and followed by DNA sequencing12,18. A large quantity of endotoxin-free plasmid DNA was prepared as described in the protocol step 2.
<ol>
	<li>Anesthetize acufected mice by intraperitoneal injection of tribromoethanol (400 &#956;g 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-15&#916;E7 plasmid DNA (10 &#956;g in 10 &#956;L PBS per dose) is acufected on 2 target sites (1 cm x 1 cm each).</li>
	<li>Mark flank skin (2 cm x 2 cm) covering the acufected area.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Euthanize mice by injecting an overdose of tribromoethanol (800 &#956;g 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).</li>
</ol>
6. Homogenate of Mouse Skin
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Quickly place the pulverized tissue to a new tube containing 500 &#956;L of cell lysis reagent for RNA extraction or to a tube containing 50 &#956;L of PBS and 1x protease inhibitor cocktail to obtain protein lysate.</li>
</ol>
7. H&#38;E and Immunohistochemical Staining of Skin Section
<ol>
	<li>Place the skin tissue in a cassette and immerse it in 4% paraformaldehyde overnight.</li>
	<li>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 &#956;m. Place tissue sections onto positively charged slides.</li>
	<li>Heat the slides at 65&#160;&#176;C for 15 min.</li>
	<li>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.</li>
	<li>Perform hematoxylin and eosin staining. Note: H &#38; E stain was performed upon request at Pathology Laboratory at the LAC, NTU.</li>
	<li>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.</li>
	<li>Dip the slide in PBS to rinse out the block solution.</li>
	<li>Dilute the block solution in PBS at 1:10 and add 2% fetal bovine serum (FBS) (diluent solution).</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Wash slides three times in TBS plus 0.1% Tween-20 (TBST solution) for 5 min each. Change solution between washes.</li>
	<li>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&#39; fragment. The reagent is ready to use for immunohistochemical staining of mouse tissue sections.</li>
	<li>Wash slides three times in TBST for 5 min each. Change solution between washes.</li>
	<li>Pipette 3,3&#39;-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.</li>
	<li>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.</li>
	<li>Place a coverslip over the stained section that is covered with permanent mounting medium (8 &#956;L per section).</li>
</ol>
8. Photograph and Analysis of Stained Tissue Section
<ol>
	<li>Visualize H &#38;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
	<ol>
		<li>Scan the whole stained sections using a scanscope with a 20X objective.</li>
		<li>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 &#38; image analysis software. Epidermal thickness is defined by the distance between the basal layer and the outermost layer of the epidermis.</li>
		<li>Count the number of Ly6G-immunoreactive cells in the dermal area (230 x 270 &#956;m2 rectangle) immediately below topical IMQ-treated skin.</li>
	</ol>
	</li>
</ol>

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<li>Tang, D. C., DeVit, M., Johnston, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+immunization+is+a+simple+method+for+eliciting+an+immune+response%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genetic immunization is a simple method for eliciting an immune response.</a> Nature. 356, 152-154 (1992).</li>
<li>Haynes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle-mediated+DNA+vaccine+delivery+to+the+skin%5BTitle%5D)+AND+%22Expert+Opin+Biol+Ther%22%5BJournal%5D)">Particle-mediated DNA vaccine delivery to the skin.</a> Expert Opin Biol Ther. 4, 889-900 (2004).</li>
<li>Tuomela, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biodistribution+and+general+safety+of+a+naked+DNA+plasmid%2C+GTU-MultiHIV%2C+in+a+rat%2C+using+a+quantitative+PCR+method%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Biodistribution and general safety of a naked DNA plasmid, GTU-MultiHIV, in a rat, using a quantitative PCR method.</a> Vaccine. 23, 890-896 (2005).</li>
<li>Eriksson, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+gene+transfer+to+skin+and+wound+by+microseeding%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">In vivo gene transfer to skin and wound by microseeding.</a> J Surg Res. 78, 85-91 (1998).</li>
<li>Lee, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+alternatively+spliced+IL-15+isoform+modulates+abrasion-induced+keratinocyte+activation%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">An alternatively spliced IL-15 isoform modulates abrasion-induced keratinocyte activation.</a> J Invest Dermatol. 135, 1329-1337 (2015).</li>
<li>Rudy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imiquimod+%28Aldara%29%3A+modifying+the+immune+response%5BTitle%5D)+AND+%22Dermatol+Nurs%22%5BJournal%5D)">Imiquimod (Aldara): modifying the immune response.</a> Dermatol Nurs. 14, 268-270 (2002).</li>
<li>van der Fits, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imiquimod-induced+psoriasis-like+skin+inflammation+in+mice+is+mediated+via+the+IL-23%2FIL-17+axis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.</a> J Immunol. 182, 5836-5845 (2009).</li>
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The authors have no financial interests to disclose.

The most critical step to ensure the expression of the acufected plasmid DNA is to evenly oscillate and loosen the horny layer of the skin. While pushing the needles gently without cutting the skin, the force should be hard enough to depress the surface. To facilitate absorption of DNA in 10 &#956;L solution on a 1 cm x 1 cm surface area, the needles should oscillate up-and-down for about 100 times in 30 s. One can determine the force that needs to prick the skin and by noting the number of times that is needed to yield ...

Skin is the first-line of host defense. Keratinocytes (KCs) are the major cell type in the skin of humans and mice. In response to environmental stimuli (e.g. sunlight, oxygen, chemicals and pathogenic invasion), KCs are activated and produce a wide array of proinflammatory cytokines and chemokines such as IL-8, IL-6, IL-1&#945;, IL-1&#946;, TNF-&#945; and GM-CSF1. Together they trigger the recruitment of immune cells to the skin. Aggravated cutaneous inflammation is often associated with numerous human diseases including acute ectopic contact dermatitis and chronic T cell-mediated inflammation (e.g. allergic contact dermatitis and psoriasis)2. Modulating proinflammatory responses in the skin by inhibiting KC activation is a plausible approach to treat cutaneous inflammation. This protocol describes a new approach to transiently express a cytokine gene in the epidermis in order to study immune response consequential to such a treatment in the skin.
The epidermis composes the most superficial layer of the skin. It serves as a physical barrier keeping external substances such as nucleic acids and pathogens from entering the deeper layers of the skin. Several needle-free techniques have been established for epidermal DNA transfer3,4. DNA in solution or associated with cationic liposomes or adenovirus vectors has been directly applied to the epidermis modified with techniques such as the removal of the stratum corneum or the treatment with depilating reagent. Removal of the cornified epithelium facilitates DNA crossing the epidermal barrier and interaction with keratinocytes and Langerhans cells to induce immune responses5. While a large surface area is available for DNA transfer and this approach does not involve needles, there are disadvantages including the requirement for the large quantities of DNA (10 - 100 &#956;g), harsh treatment on the skin by stripping, and the inconsistent results in inducing immune response in the immunized animals without DNA delivery booster6. Microparticle-mediated intracellular delivery of naked DNA to the skin has been shown to be highly efficient and reproducible for inducing immune response7,8. A hand-held gene gun has been used to bombard the skin target site with plasmid DNA-coated gold particles 2 &#956;m-diameter in size by means of pressurized helium air flow9. The plasmid DNA is presumably taken up by KCsand dendritic cells (DCs) in the skin and the protein is locally expressed or being transported to draining lymph nodes by DCs10. Although the gene gun system is simple and requires limited technical experience, the cost for preparing and delivering the &#34;DNA bullet&#34; (i.e. gold powders, helium gas) and for the gene gun itself has limited its general application. In addition, the requirement for a helium gas delivery system limits the ease of gene gun transport when the experiments are conducted at different locations. Microseeding has been demonstrated to achieve a higher efficiency of gene transfer than single injection and particle bombardment11. Microseeding uses a tattoo gun to deliver DNA to the skin without the use of particle beads11. Oscillating the microneedles on the skin using this approach is likely to directly scrape the cell membrane and thus transfer DNA to multiple cells at the same time. However, pain associated with the large number of injections with 0.254 mm-diameter needles is a disadvantage.
Here we provide an alternative approach to DNA delivery in vivo. The &#34;acufection&#34; protocol we described here provides an economical and efficient way to deliver plasmid DNA in mouse skin. Briefly, ten acupuncture needles (0.2 mm in diameter x 13 mm in length) were bound in a bundle by adhesive tape. A volume of 10 &#956;L of PBS with 10 &#956;g of plasmid DNA containing the transgene of interest was placed on the shaved, depilatory cream-treated flank skin. By using the acupuncture needle bundle to oscillate the surface (1 cm x 1 cm) of the skin, the keratins at the horny layer were loosened and the plasmid DNA applied to the surface was absorbed into the epidermis. Skin damage was monitored and found to be minimal. Expression of the transfected gene in the acufected skin was confirmed by both quantitative real-time PCR (qRT-PCR) and ELISA. The immune modulatory effects of acufection-delivered IL-15&#916;E7 on cutaneous inflammation were demonstrated using the IMQ-treated mouse model12. While acufection proves to be an easy and less demanding method for DNA delivery in vivo, the optimal amount of DNA for different genes and the times to prick the skin have to be carefully optimized to obtain reproducible results.

<table><tbody><tr><td>Accu Handy Needle</td><td>Accu</td><td>36Gx0.5</td><td>Medical device</td></tr><tr><td>Nair&lt;sup&gt;TM&lt;/sup&gt; Lotion</td><td>Church &amp;amp; Dwight</td><td>N/A</td><td>Use to remove hair</td></tr><tr><td>Shandon Xyline substitute</td><td>Thermo Fisher Scientific</td><td>6764506</td><td>Deparaffinization</td></tr><tr><td>Avertin (2,2,2-Tribromethanol)</td><td>Sigma-Aldrich</td><td>T4,840-2</td><td>Anesthesia</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>152463</td><td>Solvent for the dissolution of avertin</td></tr><tr><td>Difco&lt;sup&gt;TM&lt;/sup&gt; LB broth, Miller</td><td>BD</td><td>244620</td><td>Propagation and maintenance of &lt;em&gt;E. coli&lt;/em&gt; for molecular biology</td></tr><tr><td>Mouse IL-15/IL-15R Complex ELISA Ready-SET-Go kit</td><td>eBioscience</td><td>88-7215</td><td>Measure IL-15 protein</td></tr><tr><td>Anti-mouse Ly6G</td><td>BioLegend</td><td>127601</td><td>Antibody used for immunohistochemical stain</td></tr><tr><td>anti-mouse Ki-67</td><td>eBioscience</td><td>14-5698-80</td><td>Antibody used for immunohistochemical stain</td></tr><tr><td>Simple Stain Mouse MAXPO (Rat)</td><td>Nichirei Biosciences</td><td>414341F</td><td>Reagent to block background signal in mouse-on-mouse stain</td></tr><tr><td>DAB Peroxidase (HRP) Substrate Kit</td><td>Vector Laboratories</td><td>SK-4100</td><td>Peroxidse substrate for immunohistochemical stain</td></tr><tr><td>Ultra V block</td><td>Thermo Fisher Scientific</td><td>TA-060-UB</td><td>Reduce nonspecific background staining</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>M8884</td><td>Nuclear staining</td></tr><tr><td>Vecta Mount&lt;sup&gt;TM&lt;/sup&gt;</td><td>Vector Laboratories</td><td>M-5000</td><td>Permanently preserving histochemical stains</td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>04-693-116-001</td><td>Inhibit protease activity in cell lysate</td></tr><tr><td>Aldara cream</td><td>3M Parmaceuticals</td><td>N/A</td><td>5% imiquimod cream</td></tr><tr><td>Bessman Tissue Pulverizer</td><td>Spectrum Labs</td><td>189475</td><td>Use to pulverize skin tissue</td></tr><tr><td>ABI7900HT cycler</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>quantitative real-time PCR assay</td></tr><tr><td>Camcorder</td><td>Panasonic</td><td>HDC-SD60</td><td>Image documentation</td></tr><tr><td>Axio Scope.A1</td><td>ZEISS</td><td>N/A</td><td>Bright-field microscopy</td></tr></tbody></table>

development of an economical dna delivery system by "acufection" and its application to skin research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

While some redness was evident within the first hour after pricking with acupuncture needles, the skin cleared on day 2 and no adverse skin reaction up to day 7 was observed after pricking (Figure 2A). Needle pricking induced very low level of epidermal orthokeratosis and minimal dermal infiltration by H&#38;E analysis compared with untreated skin (Figure 2B). The epidermal thickness (Figure 2C) and ...

Watch this Scientific Journal Video about Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research at JoVE.com

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

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

development-an-economical-dna-delivery-system-acufection-its

Research

JoVE Journal

Immunology and Infection

7.4K Views.  National Taiwan University College of Medicine. This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Video: Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Genetics

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Bioengineering

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Developmental Biology

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Using Carbon Nanofiber Arrays for Efficient Biomolecule and Dye Delivery to Plants

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 &#181;L droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Author Spotlight: Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays 

Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

Medicine

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Biology

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

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

Summary

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Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys₂-His₂ Zinc-finger Domains

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