この作品は、NIHからの補助金によって支えられて：R03 DK089061-01（VKY）; NIH：K08 DK068391（VKY）、糖尿病·内分泌研究センター - （DERC - P30DK079638）ベイラー医科大学からパイロット＆フィージビリティ助成DERC（VKY）、若年性糖尿病研究財団：JDRF賞＃5-2006-134（VKY）。

<ol>
	<li>Parks, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570 (1996).</li><li>Kim, I. H., Jozkowicz, A., Piedra, P. A., Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+correction+of+genetic+deficiency+in+mice+with+a+single+injection+of+helper-dependent+adenoviral+vector.">Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13282-13287 (2001).</li><li>Belalcazar, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+expression+of+human+apolipoprotein+A-I+mediated+by+helper-dependent+adenovirus+gene+transfer+inhibits+atherosclerosis+progression+and+remodels+atherosclerotic+plaques+in+a+mouse+model+of+familial+hypercholesterolemia.">Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirus gene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia.</a> Circulation. 107, 2726-2732 (2003).</li><li>Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+correction+of+low-density+lipoprotein+receptor-deficient+mice+with+a+helper-dependent+adenoviral+vector+expressing+the+very+low-density+lipoprotein+receptor.">Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.</a> Circulation. 103, 1274-1281 (2001).</li><li>Nomura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+gene+therapy+using+helper-dependent+adenovirus+produces+long-term+protection+against+atherosclerosis+in+a+mouse+model+of+familial+hypercholesterolemia.">Low-density lipoprotein receptor gene therapy using helper-dependent adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia.</a> Gene Ther. 11, 1540-1548 (2004).</li><li>Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+Cre/loxP-based+system+for+construction+of+adenoviral+vectors.">A high-efficiency Cre/loxP-based system for construction of adenoviral vectors.</a> Hum. Gene Ther. 10, 2667-2672 (1999).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol. Ther. 8, 846-852 (2003).</li><li>Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-Dependent+Adenoviral+Vectors.">Helper-Dependent Adenoviral Vectors.</a> Current Protocols in Molecular Biology. , 16.24.1-16.24.23 (2005).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin3+is+sufficient+for+transdetermination+of+hepatic+progenitor+cells+into+neo-islets+in+vivo+but+not+transdifferentiation+of+hepatocytes.">Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.</a> Dev. Cell. 16, 358-373 (2009).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+with+Neurogenin+3+and+Betacellulin+Reverses+Major+Metabolic+Problems+in+Insulin-Deficient+Diabetic+Mice.">Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice.</a> Endocrinology. , (2009).</li></ol>

特別な利害関係は宣言されません。

HDAdsは、初期世代の広告の弱点を克服するために、遺伝子治療アプリケーション用に活用するために開発されている。しかし、技術的な課題が残っている。たとえば、HDAdはHDAdの包装とベクトルを増幅するためのHVは初期世代の広告ほど効率的ではありませんが必要です。 HVは、第一世代の広告とHV妥協HDAdの有効性の汚染である。したがって、各継代のために非常に効率的なトランスフェクションし、最適な条件が重要である。ベクトルの生産のためのもう一つの重要なパラメータは、直接浮遊細胞用種菌として使用され、その後の通路5に使用されるべきである通路（P1〜P4）です。我々の経験に、最良の結果がHDAdベクトル割合が劇的に次の一節（ 図2のP3）に増加していることで通路を使用することによって得られる。 HDAdベクターの収量は、導入遺伝子カセットに依存します。両方の遺伝子が下にあるので、ベクトル生産時には、両方の導入遺伝子が発現されるユビキタスプロモーター。 Ngn3は、転写因子とBTCは、そのことを表現成長ホルモンは、ベクトルの複製とパッケージングするのに役立ちますしながら細胞系統に影響を与える可能性の転写因子を発現HDAdベクターはベクター増幅を阻害を示唆している成長因子である。 糖尿病が大流行と想定すると、B細胞塊を復元するための新たなアプローチが必要とされている。本報告書では、膵島成長因子と共に膵島系譜定義する転写因子、Ngn3の遺伝子導入を行うためにHDAdベクトルの利点を活用する方法を記述し、ベータセルリンは、肝臓の門脈周囲の地域で膵島新生を誘導した。これの有効性を評価するためには、安定した高血糖を持つマウスを選択し、適切なコントロールが常に含まれていることを確認することが重要です。この遺伝子導入実験では、空のベクター治療糖尿病マウスは常に利用されるべきである。また、糖尿病のマイルは個別に扱わHDAd-Ngn3とHDAd-BTCを使用してceは膵島新生におけるこれら二つの遺伝子の個々の貢献をテストするために提供しています。我々のデータは、単独でNgn3、膵島新生を誘導するのに十分であることを示しているが、成長因子、BTCの添加は膵島新生の強力な誘導につながる応答を増大するのに役立つ。としてこれは、ベクトル表現が実際に標的組織、肝臓で達成され、また、処置されたマウスの血漿中でアッセイインスリンは膵臓の不在を証明することによって、膵臓の残留膵島から来ていないことを実証することをテストすることも重要である糖尿病マウスの膵島。 要約すると、遺伝子導入のためのHDAd - ベクター系の利点は、その高いクローニング能力、効率的な伝達と最小慢性毒性ならびに宿主へのベクターのゲノムの非統合のその性質を持つ肝臓で長期的な遺伝子発現にある染色体。主要な制限は、その世代とに関与する複雑な手順です。生体内のアプリケーションで主に最も人気のある広告の血清型5と肝臓に限られています。膵島新生は完全膵島成長因子、ベータセルリンと共に膵島系譜定義する転写因子、Ngn3の遺伝子導入することにより、肝臓での膵島新生を誘導することによって、糖尿病マウスにおける血漿インスリンと耐糖能を復元するように誘導することができる。本報告では、我々は高品質HDAd-Ngn3とHDAd-BTCを生成するために最適なプロトコルを示しており、高血糖症を逆に糖尿病マウスの肝臓において膵島新生を誘導し、評価するためのテクニックを紹介します。 脚注：ウイルスベクターおよびここで説明された細胞株は、ベクトルの生産コア研究所糖尿病研究センターは、ベイラー医科大学（から入手可能である<a href="http://www.bcm.edu/mcb/index.cfm?pmid=7731" target="_blank">http://www.bcm.edu/mcb/index.cfm?pmid=7731</a> ）。いくつかの市販のキットには、HDAdウイルス（ 例えば Microbix biosyst生成するためにも利用できますEMS社）。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> ProFectionR哺乳類トランスフェクションキット</td><td>プロメガ</td><td> E1200 </td></tr><tr><td> DNeasyブラッド＆ティッシュキット（50） </td><td>キアゲン</td><td> 69504 </td></tr><tr><td>パーフェクSYBR Green Supermixを、ROX </td><td>クアンタサイエンス</td><td> 95055-500 </td></tr><tr><td>デオキシコール酸ナトリウム</td><td>シグマ</td><td> D6750-25G </td></tr><tr><td> MEM粉末</td><td>インビトロジェン</td><td> 61100087 </td></tr><tr><td>ペニシリンストレプトマイシン</td><td>シグマ</td><td> 15140122 </td></tr><tr><td> FBS </td><td>アトランタバイオ</td><td> S11150 </td></tr><tr><td> L-グルタミン酸</td><td>インビトロジェン</td><td> 25030-081 </td></tr><tr><td>ハイグロマイシンB </td><td>シグマ</td><td> H0654-1G </td></tr><tR&gt; <td> MEM EAGLE JOKLIK </td><td>シグマ</td><td> M0518-10L </td></tr><tr><td> RNアーゼ</td><td>ロッシュ</td><td> 10109169001 </td></tr><tr><td> DNase Iを、グレードII </td><td>ロッシュ</td><td> 10104159001 </td></tr><tr><td>ストレプトゾシン</td><td>シグマ</td><td> s0130 </td></tr><tr><td>ガラススピナーフラスコ</td><td>コー​​ニング</td><td> 4500-3L </td></tr><tr><td>ガラススピナーフラスコ</td><td>コー​​ニング</td><td> 4500-250 </td></tr><tr><td> -アナライザcassetをスライドさせて</td><td>ピアスCH </td><td> PI66380 </td></tr><tr><td>チューブoptisealポリallomer、11.2ミリリットル</td><td>ベックマン·コールター</td><td> 362181 </td></tr><tr><td>塩化セシウム1キロ</td><td> JT4042-2 </td><td> VWR </td></tr><tr><td>ベックマンLE-80K </td><td>ベックマン·コールター</td><td>最適なLE-80K超遠心</td></tr><tr><td>フィルター</td><td> VWR </td><td> 28143-338 </td></tr><tr><td>管500メートルを遠心リットル</td><td>コー​​ニング</td><td> 431123 </td></tr><tr><td> tailveiner制止</td><td>ブレーントリーの科学的、INC </td><td> TV-150 </td></tr><tr><td>インスリン、マウスELISA </td><td> Mercodia </td><td> 10-1247-01 </td></tr><tr><td> microvette CB300 </td><td>ザルスタット</td><td> 16.443.100 </td></tr><tr><td> D-グルコース</td><td>シグマ</td><td> G8270 </td></tr><tr><td>マウスC-ペプチドELISAキット</td><td>和光純薬工業株式会社</td><td> ＃631から07231 </td></tr><tr><td>モルモット抗インスリン抗体</td><td>アブカム</td><td> ab7842 </td></tr><tr><td>ヤギ抗PDX1抗体</td><td>博士クリストファー·ライトからの贈り物</td><td></td></tr><tr><td>マウス抗Ngn3抗体</td><td>ベータ細胞生物学コンソーシアム、 東大。ペンシルベニア州の</td><td> AB2013 </td></tr><tr><td>マウス抗Nkx6.1抗体</td><td>ベータ細胞生物学コンソーシアム、 東大。 Pennsyのlvania </td><td> F64A6B4 </td></tr><tr><td>抗ベータセルリン抗体</td><td>セルサイエンス</td><td> PAAQ1 </td></tr><tr><td> ALT（SGPT）色試薬セット。 </td><td>テコ診断</td><td> A526 - 120 </td></tr><tr><td> AST /（SGOT）、カラーエンド試薬セット</td><td>テコ診断</td><td> A561-120 </td></tr></tbody></table> 表2に特異的な試薬および器具。

neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer

1型糖尿病は、膵臓のインスリン産生細胞のT細胞媒介性自己免疫破壊によって引き起こされます。膵島移植は、ドナーの可用性ごとおよび長期免疫抑制の必要性により制限されているので、今までインスリン補充は、依然として主要な治療法です。 Neuogenin3の遺伝子導入（Ngn3）によって誘導される膵島新生、膵島系譜定義する特定の転写因子とは、ベータセルリン（BTC）、膵島の成長因子は、1型糖尿病を治すために可能性を秘めています。 アデノウイルスベクター（広告）は、高効率遺伝子導入ベクターであるが、初期世代の広告は 、in vivoで使用中のため、いくつかの欠点を持っています。ヘルパー依存型広告（HDAds）は、広告の初期世代の安全性プロファイルを向上させ、導入遺伝子の発現1を延長するために開発された最も先進的な広告です。彼らはウイルスコード配列2-5を欠いているとだけ広告シス elを保持するので、彼らは慢性毒性を欠いているベクター複製とパッケージングに必要なements。これは、36キロバイトの遺伝子までのクローニングを可能にします。 このプロトコルでは、HDAd-Ngn3とHDAd-BTC生成するとSTZ誘発糖尿病マウスにこれらのベクターを提供する方法について説明します。我々の結果は、それがHDAd-Ngn3の同時注射と肝臓でHDAd-BTC誘発 &#39;ネオ小島&#39;示し、糖尿病マウスの高血糖を反転させます。

Watch this Scientific Journal Video about Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer at JoVE.com

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

我々は、ヘルパー依存性アデノウイルスベクター（HDAd）と高血糖の反転を用いた遺伝子Neurogenin3の転送（Ngn3）とベータセルリン（BTC）によりSTZにおける肝ネオ膵島形成（ストレプトゾトシン）誘発糖尿病マウスを記述します。我々の方法は、生体内伝達および長続きが遺伝子発現におけるそれらの高効率を持つヘルパー依存型アデノウイルスベクターの機能を活用できます。

ヘルパー依存型アデノウイルスベクターによる遺伝子導入による糖尿病マウスの肝臓におけるネオ·膵島形成

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

neo-islet-formation-liver-diabetic-mice-helper-dependent-adenoviral

Research

JoVE Journal

Medicine

11.7K ビュー.  Baylor College of Medicine. 我々は、ヘルパー依存性アデノウイルスベクター（HDAd）と高血糖の反転を用いた遺伝子Neurogenin3の転送（Ngn3）とベータセルリン（BTC）によりSTZにおける肝ネオ膵島形成（ストレプトゾトシン）誘発糖尿病マウスを記述します。我々の方法は、生体内伝達および長続きが遺伝子発現におけるそれらの高効率を持つヘルパー依存型アデノウイルスベクターの?...

ビデオ: Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

マウスの肝臓虚血のためにハンギング軽量システムの利用

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

前臨床モデルにおける虚血性心不全のための遺伝子導入

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

加水分解性架橋剤を介してアデノウイルスベクターテザーを使用した金属ステント表面からの血管の遺伝子導入

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

マウスにおける胆管結紮：閉塞性胆汁うっ滞による炎症性肝障害と線維症の誘発

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

Subnormothermicマシン灌流によって機能的なヒト肝臓保全と回復

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

生体内でウサギの一般的な頚動脈内皮細胞への遺伝子導入

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

糖尿病マウスの慢性創傷を作成するためのプロトコル

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

豚に自家移植用肝細胞Ex Vivoのレンチ ウイルス媒介性遺伝子治療

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

免疫不全マウスへのCBir1 TCRトランスジェニックCD4+ T細胞の養子移入による腸管炎症の誘導

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...