Name: development of an economical dna delivery system by "acufection" and its application to skin research
Uploaded: 2017-04-19T12:30:00
Description: Watch this Scientific Journal Video about Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research at JoVE.com

这项工作是由科学技术部资助项目（MOST 103-2633-B-002-002; 104-2320-B-002-048）。我们感谢博士。贝蒂吴和谢建库·李在台大，利·泽博尼在斯坦福大学和彼得·奥夫曼博士在夏威夷大学读书的手稿，尤恩·奇恩台大技术援助，以及文奇·韦博士在农业生物技术研究中心中央研究院的技术咨询。

在8只雌性小鼠 - 12周龄被用于这项研究。 C57 / BL6野生型（WT）和IL-15缺陷（IL15 - / - ）小鼠分别从国家实验动物中心（NLAC），台湾和Taconic的农场购买。 IL-15缺陷型（IL15 - / - ）和IL-15-显性（IL15 +/-和IL15 + / +）显示出1：3的比例在第二代从IL15 +/-杂合子的横。 IL-15的基因型- / -小鼠通过PCR分析证实。小鼠保持在实验动物中心（LAC），国立台湾大?...

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以确保acufected质粒DNA的表达中最关键的步骤是均匀振荡并松开皮肤的角质层。同时轻轻推针不切开皮肤，用力要够硬踏面。为了便于在上1厘米×1cm的表面积10μLDNA溶液吸收，针应当振荡在30秒为约100倍向上和向下。可以确定需要刺破皮肤上的力和通过注意的是需要得到acufected基因的最佳表达水平的次数。 修饰质粒DNA的量施加到达到表达的令人满意的程度，可能需要acufection后，...

皮肤是机体防御的第一线。角质形成细胞（KCS）在人类和小鼠的皮肤中的主要细胞类型。响应环境刺激（ 例如阳光，氧气，化学品和病原侵入），枯否细胞被活化并产生宽促炎细胞因子和趋化因子如IL-8，IL-6，IL-1α，IL-1β，TNF-α的阵列α和GM-CSF 1。他们一起引发免疫细胞在皮肤的招聘。加重皮肤炎症常常与许多人类疾病，包括急性异位接触性皮炎，慢性T细胞介导的炎症（ 例如过敏性接触性皮炎和银屑病）2相关联。通过抑制KC活化调节皮肤炎症反应是治疗皮肤炎症一个可行的办法。本协议描述了一种新方法，瞬时表达在表皮细胞因子基因，以研究免疫RESponse继发于皮肤这样的处理。 表皮组成的皮肤最表面的一层。它作为一个物理屏障保持外部物质如核酸和病原体进入皮肤的更深层这样。一些无针技术已经建立了表皮DNA转移3，4。在溶液中的DNA或与阳离子脂质体或腺病毒载体相关联的已被直接施加到与技术修饰表皮如除去角质层的或与脱毛试剂的治疗。角化上皮的去除促进DNA交叉的表皮屏障，并与角质形成细胞和朗格汉斯细胞相互作用以诱导免疫应答5。虽然大的表面积可用于DNA转移而这种做法不涉及针，也有缺点，包括要求对于大批量DNA（10 - 100微克），在皮肤上的苛刻处理通过汽提，并且在没有诱导DNA递送增压器6在免疫动物的免疫应答的结果不一致。裸DNA到皮肤的微粒介导的细胞内递送已被证明是高效的和可再现的用于诱导免疫应答7,8。一种手持式基因枪已被用于轰击皮肤目标部位用质粒DNA包被的金颗粒直径微米大小2通过加压氦气气流9的装置。质粒DNA被想必在皮肤采取了由KCsand树突细胞（DC）和蛋白质被局部表示或树突10被输送到引流淋巴结。尽管基因枪系统简单，需要有限的技术经验，为准备和提供的“DNA BUL成本让“（ 即金粉末，氦气）和基因枪本身限制了它的一般应用。另外，当实验在不同的位置进行了氦气输送系统的要求限制了便于基因枪交通工具。Microseeding已经证明，以实现基因转移比单次注射和粒子轰击11的更高的效率。Microseeding使用纹身枪无需使用微粒珠递送DNA至皮肤11。振荡上使用该方法对皮肤的微针是有可能直接刮细胞膜，从而在同一时间传送到DNA多个小区。然而，随着大量注射用0.254毫米直径针相关的疼痛是不利的。 在这里，我们提供一种替代的方法来在体内 DNA递送。该“acufection” </str翁&gt;我们在这里描述的方案提供了一种经济和有效的方式来在小鼠皮肤递送质粒DNA。简言之，将10针灸针（直径0.2mm×13毫米长）在通过胶粘带束被束缚。将10μL的PBS用含有感兴趣的转基因的质粒DNA的10微克体积置于剃毛，脱毛霜处理的侧面上的皮肤。通过使用针刺针束产生振荡的皮肤表面（1厘米×1cm）的，在角质层的角蛋白被松开，并且施加到所述表面上的质粒DNA被吸收进入表皮。皮肤损害进行监测，发现是最小的。在acufected皮肤转基因的表达通过定量实时PCR（定量RT-PCR）和ELISA证实。使用IMQ处理的小鼠模型12 acufection递送IL-15ΔE7对皮肤炎症的免疫调节效果进行了论证。虽然acufection证明是一个简单的，少DEMAnding方法用于体内 DNA递送，DNA为不同的基因和时代刺破皮肤的最佳量必须仔细优化以获得可再现的结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accu Handy Needle</td>
			<td>Accu</td>
			<td>36Gx0.5</td>
			<td>Medical device</td>
		</tr>
		<tr>
			<td>NairTM Lotion</td>
			<td>Church &#38; 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>DifcoTM LB broth, Miller</td>
			<td>BD</td>
			<td>244620</td>
			<td>Propagation and maintenance of E. coli for molecular biology</td>
		</tr>
		<tr>
			<td>QIAGEN&#9415; Plasmid Midi Kit</td>
			<td>QIAGEN</td>
			<td>12143</td>
			<td>Purification of plasmid DNA from E. coli</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 MountTM</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>
		<tr>
			<td>ScanScope&#9415; XT</td>
			<td>Aperio</td>
			<td>N/A</td>
			<td>Whole-field image scanner</td>
		</tr>
		<tr>
			<td>MetaMorph&#9415; Research Imaging</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Quantitative analysis of skin thickness and immune-reactive cells</td>
		</tr>
	</tbody>
</table>

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

在皮肤的免疫反应的失调与许多人的皮肤疾病有关。免疫相关基因直接转移到皮肤组织是一个令人着迷的方法来研究人类疾病的小鼠模型皮肤炎症的免疫调节。这里，我们提出具有成本效益的协议，它在小鼠皮肤递送裸DNA，并导致转基因表达。该方法被精压“acufection”，表示ACU穿刺介导的DNA转染 。为了执行acufection，小鼠皮肤首先用DNA注入在磷酸盐缓冲盐水（PBS），然后与针灸针束轻轻一竖以促进DNA的吸收和转染到细胞中。质粒DNA被推测采取了由角质形成细胞和皮肤中的树突细胞（DC），并表示成蛋白质。用针本身的机械刺没有造成皮肤损伤或诱导角质细胞活化。表达方式转染基因的是在皮肤在以下acufection 2天转录和翻译水平检测和维持达7天。这种acufection方法发展的主要目标是调查IL-15的先前未定义的亚型。使用这种方法，一个可变剪接IL-15同种型有部分缺失的外显子7（IL-15ΔE7）是在皮肤中表达，随后与Toll样受体7（TLR7）激动剂，咪喹莫特（IMQ）处理，以诱导炎症。 Acufection递送皮肤IL-15ΔE7抑制角质形成细胞增殖，表皮厚度和中性粒细胞募集中IMQ诱导的皮肤炎症。与在确定皮肤炎症的调节机制越来越大的兴趣，所述这里描述的协议提供了一种成本效益的和通用的替代基因枪系统或microseeding用于体内 DNA递送。这可能潜在地允许一个新颖的功能的发现基因在皮肤或调查的用于皮肤疾病的新疗法。

虽然一些发红与针灸针刺后的第一个小时之内是明显的，皮肤清除在第2天和穿刺（ 图2A）后，观察到高达至第7天无不良性皮肤反应。穿刺针与未经处理的皮肤（ 图2B）相比，H＆E分析诱导表皮正角化和最小的真皮浸润的非常低的水平。表皮厚度（ 图2C）和Ki67 +细胞（ 图2D）?...

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

这个协议是用于在小鼠皮肤中表达裸质粒DNA具有成本效益的选择。该协议的总体目标是提供免疫相关基因植入皮肤组织划定的皮肤炎症特定基因的功能作用。

通过“Acufection”和它的皮肤研究中的应用的经济型DNA递送系统的开发

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

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

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

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The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

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鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

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Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

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A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

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The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

在肠系膜淋巴管空心​​大鼠模型：应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

种植<em&gt; Heligmosomoides Polygyrus：</em&gt;免疫调节线虫寄生虫及其分泌产物

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...