Name: hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney
Uploaded: 2018-01-08T12:30:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

L.E.W. および健康の国民の協会 [R01 DK095867] キャリア開発賞から復員部の [BX002797] がサポートされており、米国心臓協会 [15GRNT25700209] サポート j. c.健康の国民の協会 [DK093660]、復員軍人援護局の部 [BX002190] およびヴァンダービルト腎臓病センター サポート M.H.W.この資料は、VA テネシー州の谷の医療系施設のリソースとサポート作業の結果です。

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+DNA+delivery+to+whole+pig+cardiac+tissue+by+coronary+sinus+retrograde+injection+employing+non-invasive+catheterization.">Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization.</a> J Gene Med. 12 (11), 920-926 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14 (2), 129-137 (2007).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney-specific+transposon-mediated+gene+transfer+in+vivo.">Kidney-specific transposon-mediated gene transfer in vivo.</a> Sci Rep. 7, 44904 (2017).</li><li>Liang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+glutathione+S-transferase+A4+accelerates+obstruction-induced+tubule+damage+and+renal+fibrosis.">Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.</a> Journal of Pathology. 228 (4), 448-458 (2012).</li><li>Liang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+insulin-like+growth+factor-1+receptor+in+endothelial+cells+against+unilateral+ureteral+obstruction-induced+renal+fibrosis.">Protective role of insulin-like growth factor-1 receptor in endothelial cells against unilateral ureteral obstruction-induced renal fibrosis.</a> Am J Pathol. 185 (5), 1234-1250 (2015).</li><li>Corridon, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+facilitate+and+monitor+expression+of+exogenous+genes+in+the+rat+kidney+using+plasmid+and+viral+vectors.">A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.</a> Am J Physiol Renal Physiol. 304 (9), F1217-F1229 (2013).</li><li>Wallace, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+kidney+volume+in+mice+with+polycystic+kidney+disease+by+magnetic+resonance+imaging.">Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.</a> Kidney Int. 73 (6), 778-781 (2008).</li><li>Woodard, L. E., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-ing+models+and+new+therapeutic+strategies.">piggyBac-ing models and new therapeutic strategies.</a> Trends Biotechnol. 33 (9), 525-533 (2015).</li><li>Yeikilis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics+based+transfection+in+normal+and+fibrotic+rats.">Hydrodynamics based transfection in normal and fibrotic rats.</a> World J Gastroenterol. 12 (38), 6149-6155 (2006).</li><li>Thalhofer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. (41), (2010).</li><li>Wooddell, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+damage+after+delivery+of+naked+plasmid+DNA+into+skeletal+muscles+is+batch+dependent.">Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.</a> Hum Gene Ther. 22 (2), 225-235 (2011).</li><li>Crespo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+liver+gene+transfer+mechanism+involves+transient+sinusoidal+blood+stasis+and+massive+hepatocyte+endocytic+vesicles.">Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.</a> Gene Ther. 12 (11), 927-935 (2005).</li><li>Rocca, C. J., Ur, S. N., Harrison, F., Cherqui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV9+combined+with+renal+vein+injection+is+optimal+for+kidney-targeted+gene+delivery:+conclusion+of+a+comparative+study.">rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.</a> Gene Ther. 21 (6), 618-628 (2014).</li></ol>

著者が開示し、利害の対立を宣言しない何もありません。

このプロトコルでは、腎臓における具体的に再現性のある遺伝子発現を達成するための手法を説明します。適度に経験豊富な外科医の手の中、我々 はマウスの年齢に応じて、50-100% の範囲で遺伝子の読み出しの感度であるためこの手法で導入したマウスの割合を発見しました。ルシフェラーゼ遺伝子の発現レベルは、トランスポゾンpiggyBacを受信マウスの数ヶ月は、背景の上、受信同?...

流体の尾静脈注入法マウス肝1,2遺伝子発現の高いレベルを達成するために一般的に使用される方法となっています。腎臓は、はるかに低いレベル、約 100 以下3でこの手法によっても transfected します。ここで説明した流体力学的腎盂インジェクションは肝4,5 以前確立されている同じ流体力学的原則を使用して物理的な手段によってオルガン式の特異性を制御する簡単な方法を提供します。、筋肉6、および他の器官7,8。このメソッドは、圧力を使用して、生きた動物体内で細胞を transfects し、同時に、すぐに臓器への損傷を誘発する細胞に含まれている DNA の流体を強制的にスピード解決9。インスリン注射器で注射と一緒に逃げ面切開10を介して腎臓を視覚化する確立された手技を使用して、さまざまな種類の腎臓の細胞、主に間質細胞の成功したトランスフェクションを発見しました。細管と収集の管11。これらのマウスの解剖は、他の臓器がイメージング テクニック11ルシフェラーゼによる可視化するのに十分高いレベルでトランスフェクションいないことを示しています。技術は非ウイルス性なので、transfection のためプラスミッド DNA の使用は注入に必要な試薬を高速かつ簡単に準備を許可します。
抗酸化物質グルタチオン S トランスフェラーゼ A4、インスリン様成長因子-1 受容体と期待される生物学的効果11,とすべての腎臓のホルモンのエリスロポエチンを表現するのにローカライズされた流体の注射を使用しています。12,13。 プロモーター選択 DNA 投与量、注入量の管理のルートの詳細な評価が実行される11をされています。さらに、両方piggyBacトランスポゾン システムおよび/またはシクロホスファミド治療遺伝子を免疫反応を抑制するためは、長期的な遺伝子発現の結果11を改善するために示されています。他の研究者は、1 ヶ月14より大きいの期間の高いトランスフェクション効率を達成する成功をラット腎静脈アプローチを使用しています。しかし、人間の病気を模倣した表現型の遺伝的補正通常実行されますマウスの最初概念実証として以来、ほとんどの哺乳類の遺伝学的モデルは、マウス モデル。我々 は腎静脈注射腎盂注入を比較し、腎盂への注入が遺伝子発現 (約 10 倍高い) と生存11腎静脈よりも優れていたことを発見します。腎盂は腎臓への参入の理想的なルートは、尿の生産の変動を許容する柔軟性が、水腎症の間に拡張した場合でもその構造の整合性を維持することがしばしばです。また、腎盂への注入は、野末注入より腎臓によって保持される目に見えないに体液が注入された腎被膜のピアスをせず腎臓へのアクセスを許可しました。他のマウスの臓器、血管以外のエントリのルートを持っていないが、腎臓の尿の空間は理想的な注射。また、腎静脈への注射は腹腔内に血液の漏れになりました。野生型マウスの腎臓の全腎容積単一の腎臓のボリュームは流体によって腎盂流体注入量とほぼ等しいので、約 0.2 cm3、磁気共鳴画像と推定されています注入 (100 μ L)15。ここで、利用可能にした腎臓の再現可能なトランスフェクションを達成するために流体力学的腎盂注入プロトコルの詳細なニュアンスのすべて。

<table><tbody><tr><td>AnaSed Xylazine</td><td>Patterson Veterinary</td><td>07-808-1947</td><td>Anesthetic - Not controlled substance</td></tr><tr><td>BD Insulin Syringe 0.5 mL 29G 1/2 Inch</td><td>Cardinal Health</td><td>309306</td><td>Required syringes</td></tr><tr><td>Buprenex</td><td>Pharmacist/Veterinarian</td><td></td><td>Analgesia - Controlled Substance</td></tr><tr><td>Dynarex Disposable Towel Drape</td><td>Thermo Fisher Scientific</td><td>19-310-671</td><td>Place over heat pad</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Use only endotoxin-free plasmid DNA</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Positive Control</td><td>Charles River</td><td>PC200</td><td>Positive control for endotoxin test</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Single Test Vial</td><td>Charles River</td><td>R13500</td><td>Endotoxin test</td></tr><tr><td>Extra Fine Micro Dissecting Scissors</td><td>Roboz Surgical Instrument</td><td>RS-5882</td><td>Surgical tool</td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouch - 9&quot;</td><td>Thermo Fisher Scientific</td><td>01-812-51</td><td>For autoclaving surgical tools</td></tr><tr><td>Gaymar Heat Pump</td><td>Paragon Medical</td><td>TP-700</td><td>Water-circulating heat pump</td></tr><tr><td>Germinator 500</td><td>Roboz Surgical Instrument</td><td>DS-401</td><td>To reuse surgical tools during surgery</td></tr><tr><td>Graefe Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5136</td><td>Surgical tool</td></tr><tr><td>Graefe Tissue Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5153</td><td>Surgical tool</td></tr><tr><td>Halsey Needle Holder, 5&quot; Length</td><td>Roboz Surgical Instrument</td><td>RS-7841</td><td>Surgical tool</td></tr><tr><td>Heat pads - 15&quot; x 21&quot; - need at least 3</td><td>Paragon Medical</td><td>TP22G</td><td>For use with Gaymar Heat Pump</td></tr><tr><td>IsoFlo (Isoflurane, USP)</td><td>Abbott Animal Health</td><td>5260-04-05</td><td>For imaging and euthanasia</td></tr><tr><td>Isotec Isoflurane Delivery System Vaporizor</td><td>Smiths Medical</td><td>VCT3K2</td><td>For imaging and euthanasia</td></tr><tr><td>Ketamine</td><td>Pharmacist/Veterinarian</td><td></td><td>Anesthetic - Controlled Substance</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td>Laboratory tissues</td></tr><tr><td>Living Image software</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr><tr><td>Luciferin</td><td>Perkin Elmer</td><td>122796</td><td>For live animal imaging</td></tr><tr><td>Nanodrop 2000</td><td>Thermo Scientific</td><td>ND-2000-US-CAN</td><td>Spectrophotometer for DNA measurement</td></tr><tr><td>Prevantics Swabs</td><td>Thermo Fisher Scientific</td><td>23-100-110</td><td>For skin surgery prep</td></tr><tr><td>Prolene 5-0 sutures Taper 30&quot;</td><td>Thermo Fisher Scientific</td><td>NC0256891</td><td>Non-absorbable sutures for skin</td></tr><tr><td>Puralube Brand Opthalmic Ointment</td><td>Patterson Veterinary</td><td>07-888-2572</td><td>To keep eyes moist during surgery</td></tr><tr><td>Trans IT - QR Hydrodynamic Delivery Solution</td><td>Mirus Bio</td><td>MIR-5240</td><td>Hydrodynamic delivery buffer for diluting DNA</td></tr><tr><td>Vicryl 5-0 Sutures J303H</td><td>Thermo Fisher Scientific</td><td>NC9816710</td><td>Absorbable sutures for muscle layer</td></tr><tr><td>Wahl Mini Arco Clipper</td><td>Med-Vet International</td><td>8787-1550</td><td>Shaver for skin prep</td></tr><tr><td>Xenogen IVIS 200</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr></tbody></table>

hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney

流体の注入は、プラスミド DNA や他の物質と様々 な組織を使ってローカル、高圧環境を作成します。流体尾静脈注射、たとえば、肝臓を導入することができます確立された方法です。この原稿では、マウス腎臓腎臓特異的遺伝子発現のプラスミド DNA の直接注入による流体力学的原則の適用について説明します。マウスを麻酔し、腎臓、腎盂に直接プラスミド DNA 含有溶液の高速注入が続いて側腹部切開によって公開されています。針は 10 秒の場所に保管し、切開部位を縫合します。次の日、ライブ動物イメージング、遺伝子発現を測定するために使用可能性があります西部のしみまたは免疫組織化学または興味の蛋白質の検出選択の遺伝子に適した他のアッセイを使用します。遺伝子発現を延長する公開されたメソッドには、遺伝子に免疫応答を抑制するトランスポゾンを介した遺伝子統合およびシクロフォスファミド治療が含まれます。

手術および注入の技術はマスターならば、主要な機器や高価な材料を必要としないを実行する簡単です。側腹部切開腎手術に新しい、安楽死を予定していくつかのマウスの 1 つの訓練日で許可するか、マウスがない回復手術この手術の最初の試行は、通常よりも長くかかる場合がありますので。また、同様の手法に精通している研究者の場合は、非常に簡単な場合が?...

Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

腎臓蛋白質の非ウイルス性発現の流体力学的腎盂注入

このプロトコルでは、具体的には腎臓における導入遺伝子発現を生成する腎盂を介してマウス腎臓にプラスミド DNA を注入する方法をについて説明します。

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

hydrodynamic-renal-pelvis-injection-for-non-viral-expression-proteins

Research

JoVE Journal

Biology

18.4K ビュー.  Tennessee Valley Healthcare System. このプロトコルでは、具体的には腎臓における導入遺伝子発現を生成する腎盂を介してマウス腎臓にプラスミド DNA を注入する方法をについて説明します。

ビデオ: Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

開発にターゲットマイクロインジェクションのテクニック<em&gt;アフリカツメガエル</em&gt;腎臓

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

発生生物学

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

急性腎障害を研究するためにゼブラフィッシュ幼生の静脈内Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

生物学

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

ラット腎臓への生物製剤の血管内デリバリー

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

医学

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

腎内注射<em&gt;大腸菌</em&gt;腎盂腎炎のラットモデル

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

マウスにおける流体力学的遺伝子送達によるタンパク質の一過性発現

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

腎動脈を介した腎臓への直接薬物送達

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

いくつかの腎臓病モデルマウスにおけるポリエチレンイミンナノ粒子を用いた外因性人工合成miRNA模倣体の腎臓への送達

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

ニワトリレンズ開発におけるタンパク質発現の研究

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

著者スポットライト:ニワトリ胚水晶体におけるRCAS(A)レトロウイルスのマイクロインジェクションによる水晶体の発生と機能の調査 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

組換えRCAS(A)レトロウイルスのニワトリ胚水晶体へのマイクロインジェクション

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

腎臓蛋白質の非ウイルス性発現の流体力学的腎盂注入

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開発にターゲットマイクロインジェクションのテクニック

急性腎障害を研究するためにゼブラフィッシュ幼生の静脈内Microinjections

ラット腎臓への生物製剤の血管内デリバリー

腎内注射

マウスにおける流体力学的遺伝子送達によるタンパク質の一過性発現

腎動脈を介した腎臓への直接薬物送達

いくつかの腎臓病モデルマウスにおけるポリエチレンイミンナノ粒子を用いた外因性人工合成miRNA模倣体の腎臓への送達

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

組換えRCAS(A)レトロウイルスのニワトリ胚水晶体へのマイクロインジェクション

組換えRCAS(A)レトロウイルスのニワトリ胚水晶体へのマイクロインジェクション