eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Procedure for Acufection
NOTE: The optimal amount of DNA and the area of the target skin surface may vary for different genes and need to be further optimized. The pricking force and number of times to loosen the horny layer of the skin also may vary depending on the skin thickness. These conditions must be carefully determined after evaluating the expression level of acufected gene transcripts by qRT-PCR.
<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 nonpharmaceutical 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)&#160;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>
2. 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>

<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>QIAGEN&#174; 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>
	</tbody>
</table>

acufection-mediated delivery of immune-related genes into mouse skin tissues

Source: Lin, Y., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55206">Development of an Economical DNA Delivery System by &#34;Acufection&#34; and its Application to Skin Research.</a> J. Vis. Exp. (2017)
The video demonstrates a method for directly transferring the interleukin-15 encoding plasmid DNA into the skin tissue of an interleukin-15 deficient mouse model through acupuncture using microneedles. These microneedles generate micropunctures in the outermost skin layer, facilitating the absorption and transfection of plasmid DNA into the epidermal cells, including keratinocytes, within the skin tissue.

<img alt="Figure 1" src="/files/ftp_upload/22256/22256_Figure1.jpg" /> 
Figure 1. Illustration of Acupuncture Needles. (A) The acupuncture needle (36 G x 0.5&#34;) is composed of a handle and a needle end. (B) Ten acupuncture needles are bound in a bundle using adhesive tapes. The length of the bundle is approximately 4 cm as measured by the ruler (lower panel).

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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Prepare an anesthetized genetically modified mouse lacking cytokine interleukin-15 expression.
Shave the dorsal flank skin and apply a hair removal cream to eliminate any remaining hair shafts, facilitating improved DNA infusion into the skin in subsequent steps.
Sterilize the dorsal skin surface and mark the target area for DNA transfer.
Apply a small drop of plasmid DNA encoding interleukin-15 to the marked surface.
Using an acupuncture needle bundle, repeatedly prick the marked skin surface.
The pricking motion of the needle bundle gently oscillates the skin surface, loosening the dead cells of the outermost skin layer.
Further, the oscillating needles scrape the plasma membranes of cells, promoting acufection, or acupuncture-mediated DNA delivery into the cells, including keratinocytes and dendritic cells.
Now, allow the mouse to recover.
Successfully transfected cells in the skin produce interleukin-15 proteins.

acufection-mediated-delivery-immune-related-genes-into-mouse-skin

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

62 Görüntüleme. Kaynak: Lin, Y., et al., "Acufection" ile Ekonomik Bir DNA Taşıyıcı Sistemin Geliştirilmesi ve Cilt Araştırmalarına Uygulanması. J. Vis. Uzm. (2017) Video, interlökin-15 kodlayan plazmit DNA'sının, mikroiğneler kullanılarak akupunktur yoluyla interlökin-15 eksikliği olan bir fare modelinin cilt dokusuna doğrudan aktarılması için bir yöntemi göstermektedir. Bu mikroiğneler, cildin en dış tabakasında mikroponksiyonlar oluşturarak, plazmit DNA'nın cilt dokusu içindeki ...

Video: Bağışıklık ile İlgili Genlerin Fare Cilt Dokularına Akupreksiyon Aracılı Verilmesi - Kavram

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Tavuk İşitsel Organ gen Transferi tarafından<em&gt; Ovo olarak</em&gt; Mikro-elektroporasyon

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

JoVE Journal

Gelişim Biyolojisi

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Elektroporasyon tarafından Kemirgen Siyatik Sinir Schwann Hücreleri için Vivo Gen Transferi

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Nörobilim

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

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

TRANSKRIPT

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