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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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 ...

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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

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Developmental Biology

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

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

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

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

Biology

Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis

Source: Jingting Li et al. <a href="https://www.jove.com/v/53280"> Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis</a>, J. Vis. Exp. (2015).
This video protocol describes a cassette-based in vitro culture technique to generate&#160;human organotypic skin using epidermal keratinocytes and devitalized human dermis. They mimic the function and physiology of their in vivo tissue counterparts.

Source: Jingting Li et al.  Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis, J. Vis. Exp. (2015).
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Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Summary

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Chapters in this video

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

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

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

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis