Name: in vivo nanovector delivery of a heart-specific microrna-sponge
Uploaded: 2018-06-15T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

我々 は生化学と分子生物学のアンソニー K. L. レオンに感謝、ブルームバーグ公衆衛生大学院、ジョンズ ・ ホプキンス大学彼の技術のためにミール-181-スポンジ構造の設計に役立ちます。我々 はまた、ポリーナ Sysa シャーと専攻、比較病理学、miRNA スポンジ配信の生体内イメージングによる彼らのテクニカル サポートにジョンズ ・ ホプキンス医療機関のキャスリーン ・ Gabrielson に感謝します。
この仕事はだった (チャールズ Steenbergen) に HL39752、NIH からの補助金によって (Samarjit Das) に米国心臓協会 14SDG18890049 から科学者の開発助成金でサポートされています。ラット心臓固有のプロモーターは、シンシナティ小児病院のジェフリー D. Molkentin によって提供された寛大。

すべての実験手順は、機関動物ケアおよび使用委員会のジョンズ ・ ホプキンス大学によって承認されました。
1. スポンジ デザイン
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マイクロ Rna の 3' UTR のバインド 注: A miRNA 機能そのターゲット Mrna の 3' 非翻訳領域 (UTR) 部分的に補足のサイトと特定の基本ペアリング対話を通じて包括的なレビューを参照してくださいバーテル)15</su...

<ol>
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この記事は設計および miRNA スポンジの合成を説明し、どのようにスポンジの組織特異的発現は組織特異 miRNA 家族式を抑制する強力なツール。
我々 は心臓におけるプロモーターと発現プラスミドにスポンジをターゲット ミール 181 家族で複製することができますを示しています。配信の nanovector 粒子にプラスミッドを効率的にパッケージできる両方体外と体内<...

最後の 20 年間、人間の病気の miRNAs の重要な役割を指摘している数多くの研究がずっとあります。文献の大きいボディからの調査結果は、がん1および心血管疾患2,3,4,5などの疾患の病態において Mirna の紛れもない重要性を示しています。たとえば、ミール 21 は、増加細胞周期と細胞増殖6の結果、多くのがんで亢進です。C 型肝炎の感染症でミール 1227ウイルスの複製に重要な役割を果たしているし、ミール 122 の抑制が8ウイルス量を減少させることが示されています。心臓肥大のミール-212/132 は心で亢進、9病理学的表現型に関与します。誘導 miRNA の機能抑制または、ダウンレギュレーションの明白な重要性は、治療上ほとんどすべての病気の miRNA の生物学を悪用するための機会を示唆しています。
4 ミール 181 家族のメンバー、ミール-181a/b/c/d は、人間のゲノム ゲノム 3 つの場所にあります。非コーディングの RNA ホスト遺伝子 (MIR181A1 HG) のイントロン領域は、ミール-181-a/b - 1 クラスターをエンコードします。NR6A1 遺伝子のイントロン領域は、ミール-181-a/b-2 をエンコードします。ミール-181-c/d クラスターは染色体 19 の未同定トラン スクリプトにあります。ミール 181 家族のすべてのメンバーは同じ「シード」シーケンスを共有し、4 ミール 181 家族全員同じ mRNA ターゲットを調整できる可能性があります。
私たち3,4等10末期心不全中ミール 181 家族の重要性を強調しています。我々 はまた、ミール-181 c アップレギュレーションが II 型糖尿病など、心臓病のリスクの増加、肥満、加齢3,4、5と関連付けられる病理学の条件の下で発生することを認識しています。それは、過剰発現のミール-181 c が心臓機能障害4につながる酸化ストレスを引き起こすことが仮定されています。
いくつかのグループが、ミルナは、ミトコンドリア11,12,13,14, しかし、我々 はミール-181 c は処理、核ゲノムに由来を示すために最初に示唆しているとその後 RISC3内のミトコンドリアへの移行。さらに、ハート5のミトコンドリアのコンパートメントにミール 181a およびミール 181b 低発現が検出されました。重要なは、我々 を発見したミール-181 c mt COX1 mRNA の発現を抑制する実証 Mirna がミトコンドリア遺伝子に参加し、ミトコンドリアの機能3,4を変更することにより。
この資料では、心筋全体のミール 181 家族をノックダウンする miRNA スポンジを設計するために必要な方法論について説明します。さらに、我々 は、ミール-181-スポンジの生体内でのアプリケーションのためのプロトコルを概説します。

<table>
	<tbody>
		<tr>
			<td>pEGFP-C1 vector</td>
			<td>Addgene</td>
			<td>6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>In-fusion</td>
			<td>Clontech</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Miniprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>miR-181-sponge synthesis</td>
			<td>Introgen GeneArt</td>
			<td>custome made</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custome</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid DNA Ligation Kit</td>
			<td>Sigma-Aldrich</td>
			<td>11635379001</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>ATCC</td>
			<td>CRL-1446</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector Kits for H9c2 (2-1)</td>
			<td>Lonza</td>
			<td>VCA-1005</td>
			<td></td>
		</tr>
		<tr>
			<td>G418, Geneticin</td>
			<td>Thermo Fisher Scientific</td>
			<td>11811023</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria II Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 450 sonifier</td>
			<td>Marshall Scientific</td>
			<td>EDP 100-214-239</td>
			<td></td>
		</tr>
		<tr>
			<td>The Xenogen IVIS Spectrum optical imaging device</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MTCO1 antibody</td>
			<td>Abcam</td>
			<td>ab14705</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequoia C256 ultrasound system</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo nanovector delivery of a heart-specific microrna-sponge

マイクロ Rna (miRNA) は小さい非コーディングの RNA 転写後のメッセンジャー RNA (mRNA) の発現を抑制します。がんや循環器病などのひと疾患は、組織および/または病気の進行に関連付けられているセル固有の miRNA の表現を有効に示されています。MiRNA の発現の阻害では、治療的介入の可能性を提供しています。ただし、antagomir オリゴヌクレオチドを用いた Mirna を抑制する従来のアプローチは、グローバル配信時に特定のマイクロ Rna の機能を影響します。ここで、ラットのモデル ミール 181 家族の生体内で心臓固有抑制のためのプロトコルを提案する.MiRNA スポンジ構造 10 繰り返しアンチ miR 181 バインド シーケンスを含むように設計されています。心臓固有 α-MHC プロモーターが心臓固有ミール 181 miRNA スポンジ式を運転する pEGFP のバックボーンに複製されます。安定したセルを作成するには、ミール-181-スポンジ、筋芽細胞 H9c2 細胞を表現するラインを発現する α-MHC-EGFP-miR-181-sponge コンストラクトと蛍光活性化セル ネオマイシンと培養される GFP 陽性 H9c2 細胞に (FACs) の並べ替え順(G418)。ネオマイシンの安定的な成長は、次モノクローナル細胞集団は追加 FACs と単一細胞のクローニングによって確立されます。結果筋 H9c2-ミール-181-スポンジ-GFP の細胞は、ミール-181 ターゲット蛋白質の発現増加を評価したミール 181 家族の機能の損失を展示し、スクランブル非機能的なスポンジを発現する H9c2 細胞と比較しています。さらに、正荷電リポソーム粒子と負荷電のミール-181-スポンジ プラスミドは錯形成によってミール-181-スポンジ構造の全身の配信のための nanovector を開発します。GFP のin vivoイメージングを 3 週間にわたって、nanovector の複数の尾静脈注射は心臓に固有の方法でミール 181 スポンジの重要な表現を促進することが明らかに。重要なは、腎臓や肝臓ではなく心臓の組織で、ミール 181 機能の損失が観察されます。MiRNA のスポンジは、組織固有の miRNA の発現を抑制する強力な方法です。組織固有のプロモーターからミルナ スポンジ式の運転 miRNA 阻害は、対象となる臓器や組織に限定することができますの特異性を提供します。さらに、nanovector や miRNA スポンジの技術を組み合わせることにより、効果的な配信と組織固有の miRNA 阻害体内。

固定して transfected pEGFP-ミール-181-スポンジ-表現 H9c2 細胞 (4.2 の手順) から、全体のミール 181 家族 (ミール 181a、ミール 181b、ミール-181 c、およびミール-181 d) の式は pEGFP スクランブル表現 H9c2 細胞を基準にして緩やかな減少しました。ミール 181 家族の競争力阻害剤としてミール-181-スポンジがあり、我々 が予想する式のミール-181 c ミトコンドリア遺伝子をターゲッ?...

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

生体内で心臓特定のマイクロ Rna スポンジの Nanovector 配信

組織特異的マイクロ Rna 阻害は、マイクロ Rna において発展途上にある技術です。ここで、正常に心の底から筋細胞のマイクロ Rna ミール 181 家族を阻害するプロトコルについて述べる。Nanovector 技術を使用して、重要な生体内で心臓固有ミール 181 家族阻害を示しますスポンジ、マイクロ Rna を提供します。

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

So for the last couple of decades, the importance of microRNA has been recognized in human disease conditions, but the problem is that, therapy-wise, it did not go to the primetime. One of the problem is, one microRNA can have different targets in different tissues. So one microRNA can be protected in one organ but detrimental to the others.So this method can help to eliminate tissue-specific microRNA knockdown strategy. The main advantage of this technology is we will use a microRNA sponge delivery and can be performed a tissue-specific manner. Begin this procedure with sponge design as described in the text protocol.To remove the CMV promoter from the commercially-available EGFR plasmid, first perform a digestion reaction. Following digestion, add five times the reaction volume of column binding buffer. Purify the digested linearized plasmid with a DNA purification column as described by the manufacturer.Elute with 25 microliters of elution buffer added carefully to the center column. Gel-purify the digested plasmid on a 0.5%agarose gel by first adding five microliters of loading buffer to the eluded plasmid. Load the entire sample into one well on a 0.5%agarose gel.Include a separate lane with 500 nanograms of uncut vector. Run it for 30 to 40 minutes, until an adequate separation of cut and uncut plasmids is achieved. After cloning the cardio-specific promoter alpha-myosin heavy chain as subscribed in the text protocol, add five times the reaction volume of the column binding buffer to the PCR reaction and purify it with a DNA purification column as described by the manufacturer.Elute the mixture with 25 microliters of elution buffer added carefully to the center of the column. Add five microliters of loading buffer to the eluded plasmid and load the entire sample into one well of a 1%agarose gel. Run the gel for 30 to 40 minutes before excising the PCR product as before.Ligate the alpha-myocin heavy chain promoter with the EGFP plasmid at 50 degrees Celsius for 15 minutes before cooling the reaction on ice. Perform a bacterial transformation by transfecting 50 microliters of competent cells with 2.5 microliters of infusion ligation mix. Rest the Eppendorf tube on ice for 20 minutes.Then, incubate the reaction in a 42 degree Celsius water bath for 40 seconds and place the tube on ice for two minutes. Following the transformation, add 350 microliters of SOC media and grow the cells at 37 degrees Celsius for 45 minutes. Plate 200 microliters of the outgrowth solution on LB plates with Kanamycin.Perform a colony PCR to identify Kanamycin-resistant cells, which contain the alpha-myosin heavy chain promoter ligation. Ligate the microRNA 181 sponge into the alpha-myosin heavy chain expressed EGFP vector by setting up a 21 microliter ligation reaction as detailed in the text protocol. Perform the ligation at room temperature for five minutes and then place it on ice.Transform 50 microliters of XL1-Blue competent cells with two microliters of ligation mix as before. Perform a colony PCR to identify Kanamycin-resistant bacteria, which contain the correct plasmid cloned with the first alpha-myacin heavy chain followed by the microRNA 181 sponge construct sequence. Design screening primers to PCR across the ligation junction.Expand the PCR positive clones in LB Kanamycin broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform DNA sequencing to verify that the plasmid is expressing the desired DNA sequences. Using an electroporator, transfect rat myoblasts with either a scramble sponge as a control, or the plasmid cloned with the first alpha-myocin heavy chain, followed by the microRNA 181 sponge construct.Briefly, transfect 0.4 million cells with two microgams of plasmid DNA and 100 microliters of a solution compatible with the electroporation device. Use an appropriate program to transfect the H9C2 cells. Plate only the GFP-expressed cells into a six-well plate with an addition of neomycin to the complete growth media.After seeding five to 10 cells in each well of a 96-well plate, followed by flow sorting, expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Prepare the liposomal nanoparticles by dissolving the components listed in the text protocol in a mixture of chloroform and methanol in a glass vial. Prepare the mixture by adding 5%glucose to the vacuum-dried lipid film before leaving it overnight to allow this mixture to hydrate the film.Gently rock the vial for two to three minutes at room temperature in order to produce multilamellar vesicles. Then, incubate the vesicles with occasional shaking in a 45 degree Celsius incubator. Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for three to four minutes, until clarity can be observed.Add a 100%duty cycle and 25 watts of output power. Mix either a scramble sequence or a microRNA 181 sponge sequence cloned into the alpha-myocin heavy chain expressed EGFP plasmid with liposome on a one-to-three charge ratio basis to form the nanovector. The results of optimized three-week treatment protocols with six intravenous injections through the tail vein are shown here.The yellow colorization in the epifluorescence image demonstrates expression of the EGFP vector. The maximum intensity of yellow colorization is observed at the week three time point. The alpha-myosin heavy chain promoter sequence in the EGFP vector selectively expresses either microRNA 181 sponge or scrambled sequence in the heart at Day 21 as revealed by the yellow fluorescence.The Western blot and band densitometry showed a significant increase in the mt-COX1 expression in the microRNA 181 sponge nanovector injected rats as compared to the scramble nanovector groups, with alpha-Tubulin as the normalization control. One of the major challenges in this particular protocol is to keep the orientation of the sequencing, you need directional. Once a bacterial preparation is made, with a proper orientation of the microRNA sponge, this technique can be done with a two or three minute tail vein injection time.While attempting this procedure, it is important to sequence your bacterial colony multiple times, multiple batches, to make sure the microRNA response sequence is in the correct order. After watching this video, you should have a good understanding of how to design a microRNA sponge to knock down the expression of any microRNA family and prepare an in vivo application of microRNA sponge. Following this procedure, other methods, such as in vivo antagomir treatment can be performed to answer additional questions like whether a specific microRNA can be knocked down in all the organ at the same time.After its development, this particular technology paved the way for researcher in the field of microRNA to explore tissue-specific knockdown of a microRNA in the pathophysiology of multiple-disease conditions. Tissue-specific microRNA inhibition is currently an undeveloped technique in the microRNA field. The importance of mat-down regulation of functional inhibition of up-regulated microRNAs in human disease suggest exploring microRNA biology to create a cellular vulnerability for therapeutic intervention.

Research

JoVE Journal

Biology

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

神経刺激剤の徐放のためのMicroinfusionシステムの構築と注入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

生物学

In vivo Imaging of Deep Cortical Layers using a Microprism

In vivo Imaging of Deep Cortical Layers using a Microprism

マイクロプリズムを使ってDeep皮質層のin vivoイメージング

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

齧歯類精巣上体精子のリン酸化ペプチドの分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNAiのトリガ配信へ<em&gt;ハマダラカ</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

牛膝剥離植物における軟骨改造のEx Vivo組織培養モデル

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

In Vivo Surface Electrocardiography for Adult Zebrafish

成体ゼブラフィッシュの生体内心電図検査

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

近赤外感受性コアシェルワクチンデリバリープラットフォームの製造

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...