We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

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The authors have nothing to disclose.

この記事では、細部SBを使用したGBMの新たなモデルを生成するための汎用性と再現性のある方法は、新生児マウスの脳室下帯を周囲の細胞に発癌性プラスミドDNAの仲介統合をtransposase-。我々は、新たな注目遺伝子を有するトランスポゾンプラスミドを作製するためのプロトコルを提示し、生きた動物における腫瘍の進行を監視する方法、およびこれらの腫瘍の組織病理学的および免疫組織化?...

多形性膠芽腫（GBM）は、手術、放射線療法、および化学療法1で処理された15-21ヶ月の生存期間中央値で、成人における最も一般的な（60％）の原発性脳腫瘍である。 GBMのための新規治療法は不可欠である。実験的治療は十分にヒトの疾患の顕著な特徴を再現する動物モデルでのテストが必要。げっ歯類でGBMを誘導するための戦略は、神経膠腫細胞株2を用いた化学アルキル化剤誘発、生殖細胞または体細胞の遺伝的変化、または移植を含む。最も一般的に使用されるモデルは、脳内に、または皮下に同一の遺伝子型を有する動物における同系細胞を使用して、いずれかの同所、神経膠腫細胞株の注入を用いる。または異種細胞は、免疫に移植最も一般的ヒトGBM細胞株は、マウス3を損なわ。再現性の利便性、standa：異種移植片は、頭蓋内腫瘍の研究のための多くの利点を提供rdized成長率、死亡および腫瘍局在の時間。しかし、これらのモデルが原因注入し、正確に組織学的に再現する限られた能力のために使用される人工、侵襲的な外科的アプローチに限界がある人間のGBM（WHOグレードIV）の特性を備えています：擬似pallisading壊死、核atypias、びまん性浸潤、微小血管増殖とglomeruloid血管異常4-7の形成。ウイルスベクター8-12と、またはトランスポゾン媒介インテグレーション13のいずれかで、発癌性DNAでの体細胞のゲノムを変更することによってGBMの誘導は、より厳密にヒト疾患の病因を再現し、ヒトGBMの組織病 ​​理学的特徴を再現する。 歴史的には、CNSの腫瘍は生存14の予測因子となることが観察された起源の感知セルに基づいて分類されている。 GBMのは、プライマリとセカンダリに分類される。新たな証拠のTODAY主要神経膠芽15の非常に不均一な性質を指す。二次GBM（15％）、低悪性度星状細胞腫（WHOグレードI）およびanaplasic星状細胞腫（WHOグレードII）の悪性形質転換の結果は、疾患の初期発症、予後良好および遺伝子発現の「前神経」パターンに関連付けられている、原発性GBM（85％）のに対し、後期発症、予後不良とグリア（古典）、神経または間葉発現パターンを示している。遺伝子発現のこれらのパターンは、腫瘍の起源の実際のセルと相関するかどうかはまだ活発に研究されている。データを蓄積することのGBMに関連する遺伝子変異の組み合わせは生存のための予測であることを示しています。例えば、染色体1P / 19Qのヘテロ接合の消失（LOH）は、IDH1変異、PDGFRα増幅は、NFは、EGFRの過剰発現、Notchおよびソニックヘッジホッグ経路の活性化のに対し、二次性GBM、前神経発現パターンおよび良好な予後と関連している1およびPTEN損失およびp53の変異は、神経、古典的な、または間葉原発性GBMと悪い予後16,17と相関している。大規模な配列決定プロジェクトの出現とテストのために利用可能な多数の患者検体の蓄積は、GBMの病因および進行および治療法を具体的に合わせて調整することができる個別化医療の可能性に関与する遺伝子変異および経路に関して新しい情報を豊富にもたらし患者の遺伝的異常。最終的に、体系的な方法でこれらの突然変異および経路の予測値を評価するために、それぞれの場合において可能な治療を試験するために、予め決められた遺伝的変化でGBMの動物モデルを必要とする。ゲノムDNAのトランスポゾン仲介統合が実現可能なアプローチを提供しています。 眠れる森の美女トランスポゾンシステム、トランスポゾンのTc1が/ マリナークラスのメンバは、マルチステップproceで（構築）「覚醒」した休眠より10万年前に18,19となったサケトランスポザーゼ遺伝子、からサ ​​イト特異的変異のSS。本質的には、特定の配列（逆方向反復/ダイレクトリピート：IR / DR）に挟まれたDNAトランスポゾンは、眠れる森の美女トランスポザーゼの活動によって、「カット＆ペースト」の方法でゲノムに統合することができます。トランスポザーゼは、IRサイトの両端を認識しトランスポゾンを切除および塩基TとA、転置（ 図1a）の間にトランスポゾンの各末端に複製された塩基の間に別のDNA部位にランダムにそれを統合します。眠れる森の美女トランスポザーゼは、3つのドメイン、トランスポゾン結合ドメイン、核局在化配列および触媒ドメインから構成されている。四つトランスポザーゼ分子が一緒にトランスポゾンの両端をもたらし、転位を可能にするために必要とされるトランスポゼースのあまりに多くの分子が存在する場合、しかし、それらは二量体化を阻害するとtetramerizeでき転位反応20。効率的な転位反応は、トランスポゾンのトランスポザーゼの最適な比率を必要とする。トランスポザーゼをコードするDNAは、（シス）トランスポゾンと同じプラスミド上または（トランスで）別のプラスミド上に供給することができる。トランスポザーゼ及びトランスポゾンの間の最適な比率を確保するために、十分な活性を有するプロモーターは、（「シス」モデルの場合）トランスポザーゼの発現のために選択することができるか、注射液中のプラスミドの比は、のために（最適化することができる「トランス」モデル）。眠れる森の美女トランスポゾンシステムは、機能ゲノミクス、挿入突然変異、遺伝子導入および体細胞遺伝子治療の21のために成功裏に使用することができます。合成構築物は休眠サケバリアントから機能性分子に再設計され、眠れる森の美女トランスポザーゼは、ヒトまたは他の哺乳動物20内の他のトランスポゾンに結合しない。その発見以来、分子工学はenhancを持ってpT2をトランスポゾンで、その結果、IR配列及びトランスポゾンに隣接するTATAジヌクレオチドの追加の変化を通じてSBトランスポゾンシステムの転置有効性を編。これらのトランスポゾンは、IRサイトにSBの結合を最適化し、切除の有効性を増加している。 SBトランスポザーゼも大幅な改善を行った。本明細書に提示した実験で使用されるトランスポザーゼSB100X、哺乳動物細胞22の大規模遺伝子スクリーニングに続いてDNAシャッフリング戦略によって生成された機能亢進性トランスポザーゼである。 本報告書では、新生仔マウス23のサブ脳室帯の細胞へのプラスミドDNAの非ウイルス、トランスポゾン媒介統合を有するマウスにおいて本質的なGBMを誘導するために、迅速な汎用性と再現性のある方法を提示する。私たちは、眠れる森の美女のトランスポザーゼを用いてゲノム統合のための従順な新規遺伝子とトランスポゾンを作成し、プラスミドの取り込みを監視する方法を実証するための簡単​​な方法を提示し、生物発光を用いて、疾患の進行。我々はまた、このモデルで再現GBMの組織学的および免疫組織化学的な特徴を特徴づける。さらに、我々は、これらの腫瘍からの一次GBM細胞株を生成するために迅速な方法を提示する。腫瘍が動物に元の細胞から誘導されている眠れる森の美女モデルは、誘導及び腫瘍の進行中の候補GBM遺伝子の役割の機能的評価を可能にする。このシステムは、局所微小環境を変化させることができる侵襲性外科手術手順を必要とせず、免疫適格マウスにおける免疫療法を含めた新規GBM治療の試験のためにも適している。

<table border="0" cellpadding="0" cellspacing="0" width="952">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Stoelting&#39;s Lab Standard with Rat and Mouse Adaptors</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">51670</td>
			<td style="width:267px;">Other companies produce similar frames, any of them with small mouse adaptors are suitable&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">Quintessential Stereotaxic Injector (QSI)</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">53311</td>
			<td style="width:267px;">Injections can be made without it, ,but&#160;&#160;&#160; the automatic injector allows for increased reproducibility and convenience</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">10 &#956;L 700 series hand fitted MICROLITER syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">Model 701</td>
			<td style="width:267px;">This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:367px;">30 gauge gauge hypodermic needle 
			(1.25&#34;, 15o bevel)&#160;</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">S/O# 197462</td>
			<td style="width:267px;">This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#39;t easily enter the skin), it needs to be replaced.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">in vivo-jetPEI</td>
			<td style="width:199px;">Polyplus Transfection</td>
			<td style="width:119px;">201-10G</td>
			<td style="width:267px;">Aliquot in small volumes and keep at -20oC&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">D-Luciferin, Potassium Salt</td>
			<td style="width:199px;">Goldbio.com</td>
			<td style="width:119px;">LuckK-1g</td>
			<td style="width:267px;">Other sources of firefly lucifierase are just as adequate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Hematoxylin Solution, Harris Modified</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:119px;">HHS128-4L</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">62550-01</td>
			<td style="width:267px;">for embedding brains for cryosectioning and immunohistovhemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Advanced DMEM/F-12, no glutamine</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td style="width:267px;">for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">B-27&#168; Supplement (50X), serum free&#160;</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">N2 Supplement (100x)</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Normocyn</td>
			<td style="width:199px;">InvivoGen</td>
			<td style="width:119px;">ant-nr-1</td>
			<td style="width:267px;">anti-mycoplasma agent for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human EGF</td>
			<td style="width:199px;">PEPROTECH</td>
			<td style="width:119px;">AF-100-15</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human FGF-basic</td>
			<td style="width:199px;">PEPROTECH</td>
			<td>100-18B</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">HyClone HyQTase&#160; Cell Detachment Reagent&#160;</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:119px;">SV3003001</td>
			<td style="width:267px;">for the dissociation of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimble-Chase Kontes Pellet Pestle</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>K749510-0590</td>
			<td style="width:267px;">for the dissociation of freshly dissected tumors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Falcon Cell Strainers mesh size 70um</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:267px;">for generating single cell suspensions of dissociated neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Xylenes Histological grade</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">C8H10</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Protocol Harris Hematoxylin Mercury free ( acidified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">245-678</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Protocol Eosyn Y Solution ( intensified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">314-631</td>
			<td style="width:267px;"></td>
		</tr>
	</tbody>
</table>

transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

SB-誘発性神経膠芽の組織病 ​​理学的特徴を特徴づけるために、C57 / BL6新生児マウスは、発癌性DNAとトランスポゾンをコードするプラスミドと組み合わせてルシフェラーゼをコードするプラスミド（pT2を/ SB100xリュック）とP1で注入した、 すなわち 、NRAS（PT / CAGGS NRAS（ 図3d）との組み合わせで-NRASV12）およびSV40 LGT（PT / CMVSV40-LGT）（ 図3c）またはPDGFβと短い?...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

新生児マウスの脳室下帯へのプラスミドDNAのトランスポゾン媒介統合は、神経膠芽腫の新規モデルを生成する

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

transposon-mediated-integration-plasmid-dna-into-subventricular-zone

Research

JoVE Journal

Medicine

12.8K ビュー.  University of Michigan School of Medicine. Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

ビデオ: Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

新生児低酸素症の生化学的測定

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

医学

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

新しいハーブ治療法を評価するために敗血症の動物モデルの使用

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

の同定<em&gt;眠れる森の美女</emリンカ媒介PCRを用いた固形腫瘍&gt;トランスポゾン挿入

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

脳のアガロースゲルモデルに画像誘導対流増加送達

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

マウス腎臓移植：同種移植片拒絶のモデル

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生児低酸素性虚血性脳症の子豚モデル

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

マウスを用いた胃食道逆流の外科モデル

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

新生児マウスの心筋梗塞、心臓再生のモデル

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...