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.

In diesem Artikel werden wir ausführlich ein vielseitiges und reproduzierbares Verfahren zur Erzeugung von neuen Modellen der GBM mit SB transposase- vermittelte Integration onkogenen Plasmid-DNA in die Zellen umgebenden Subventrikularzone von neugeborenen Mäusen. Wir stellen ein Protokoll zur Transposon-Plasmiden mit neuen Genen von Interesse zu erzeugen, zu veranschaulichen, wie das Fortschreiten von Tumoren in lebenden Tieren zu überwachen und, wie histopathologischen und immunhistochemischen Eigenschaften dieser ...

Glioblastoma multiforme (GBM) ist die häufigste (60%) primäre Hirntumoren bei Erwachsenen, mit einer medianen Überlebenszeit von 15 bis 21 Monaten, wenn sie mit Operation, Bestrahlung und Chemotherapie 1 behandelt. Neuartige Therapien für GBM sind unerlässlich. Experimentelle Therapien erfordern Tests an Tiermodellen, die die wesentlichen Merkmale der menschlichen Krankheit angemessen zu reproduzieren. Strategien zur GBM induzieren bei Nagetieren sind chemische Mutagenese mit alkylierenden Substanzen, Keimbahn oder somatischen genetischen Veränderungen, oder Transplantation mit Gliomzelllinien 2. Die am häufigsten verwendeten Modelle verwenden die Implantation Gliomzelllinien entweder orthotopisch, in das Gehirn oder subkutan mit syngenen Zellen in Tieren, die mit identischen Genotyp; oder xenogene Zellen, am häufigsten menschlichen GBM-Zelllinien, in Immun implantiert beeinträchtigt Mäusen 3. Xenografts bieten viele Vorteile für die Untersuchung der intrakraniellen Tumoren: Bequemlichkeit der Reproduzierbarkeit, standardized Wachstumsraten, Zeit des Todes und der Tumorlokalisation. Allerdings haben diese Modelle Einschränkungen auf Grund der künstlichen, invasive chirurgische Ansatz für Implantationen und begrenzte Fähigkeit, histologische genau zu reproduzieren verwendet charakteristischen Merkmale der menschlichen GBM (WHO Grad IV): Pseudo pallisading Nekrose, Kern Atypien, diffuse Invasion, Mikrogefäßproliferations und die Bildung von glomeruloid Gefäßveränderungen 4-7. Induktion der GBM durch Verändern des Genoms von somatischen Zellen mit onkogener DNA, entweder mit viralen Vektoren 8-12 oder mit Transposon-vermittelte Integration 13 reproduziert enger die Ätiologie der Krankheit beim Menschen und rekapituliert histopathologischen Merkmale der menschlichen GBM. Historisch gesehen, Tumoren des ZNS wurden klassifiziert, basierend auf der wahrgenommenen Ursprungszelle, die beobachtet wurde, würde ein prädiktiver Faktor für das Überleben 14 sein. GBM sind in primäre und sekundäre eingestuft. Anzeichen dafür, today weist auf die sehr heterogenen Natur der primären Glioblastomen 15. Sekundäre GBM (15%), das Ergebnis der malignen Transformation von Astrozytomen niedrig (WHO Grad I) und anaplasic Astrozytome (WHO Grad II) werden mit früheren Ausbruch der Krankheit, bessere Prognose und einem &quot;proneurale&quot; Muster der Genexpression assoziiert , während primäre GBM (85%) zeigen einen späten Beginn, schlechte Prognose und Gliazellen (klassisch), neuronale oder mesenchymalen Expressionsmuster. Ob diese Muster der Genexpression korrelieren mit der eigentlichen Ursprungszelle des Tumors noch intensiv erforscht. Sammeln von Daten zeigt, dass die Kombination von genetischen Mutationen, die mit GBMs zugehörige prädiktive Überleben sind. Beispielsweise werden Verlust Heterozygotie (LOH) der Chromosomen 1p / 19q, IDH1 Mutationen, PDGFRα Amplifikationen mit sekundären GBMs, proneurale Expressionsmuster und bessere Prognose assoziiert, während EGFR-Überexpression, Notch und Sonic Hedgehog-Pfad aktiviert, Nf1 und PTEN Verlust und Mutationen von p53 mit neuronalen, Klassik, oder mesenchymale primären GBM und schlechtere Prognose 16,17 korreliert. Das Aufkommen der Großsequenzierungsprojekte und die Ansammlung von zahlreichen Patientenproben zum Testen zur Verfügung bringt eine Fülle von neuen Informationen in Bezug auf genetische Mutationen und Wege in GBM Pathogenese und Progression und die Möglichkeit der individualisierten Medizin, wo Therapien gezielt auf zugeschnitten werden verwickelt die genetische Anomalien des Patienten. Letztlich, um die Aussagekraft dieser Mutationen und Wege in systematischer Weise zu bewerten und mögliche Behandlungen jeweils zu testen, benötigt Tiermodellen der GBM mit vorgegebenen genetischen Veränderungen. Transposon-vermittelte Integration genomischer DNA bietet ein gangbarer Weg. Das Transposon Sleeping Beauty Systems, Mitglied des Tc1 / mariner Klasse von Transposons, wurde &quot;erwacht&quot; (gebaut) in einem mehrstufigen Verfahss von ortsspezifische Mutagenese von einer Lachs Transposasegen, die vor mehr als ruhend 10.000.000 Jahre 18,19 wurde. Im Wesentlichen DNA Transposons durch spezifische Sequenzen (inverted repeats / direkte Wiederholungen: IR / DR) flankiert kann in das Genom in einem &quot;cut and paste&quot; Weise durch die Aktivität des Dornröschen Transposase integriert werden. Die Transposase erkennt die Enden der IR Sites, schneidet das Transposon und integriert sie zufällig in einem anderen DNA-Region zwischen den Basen T und A, Basen, die an jedem Ende des Transposons während der Umsetzung (Abbildung 1a) dupliziert werden. Dornröschen Transposase wird aus drei Domänen, ein Transposon-bindende Domäne, eine Kernlokalisationssequenz und eine katalytische Domäne umfasst. Vier Transposase Moleküle sind erforderlich, um die beiden Enden des Transposons zusammen zu bringen und lassen für die Umsetzung ist jedoch, wenn zu viele Moleküle des Transposase vorhanden sind, sie dimerisieren können und tetramerize zu hemmendie Umsetzung Reaktions 20. Eine effiziente Transpositionsreaktion benötigt eine optimale Verhältnis von Transposase Transposons. Die DNA, die Transposase kodiert, kann auf demselben Plasmid mit dem Transposon (in cis) oder auf einem anderen Plasmid (in trans) geliefert werden. Um das optimale Verhältnis zwischen der Transposase und Transposons zu gewährleisten, kann ein Promotor mit ausreichender Aktivität für die Expression der Transposase (für die &quot;cis&quot; Modell) oder das Verhältnis der Plasmide in der Injektionslösung kann optimiert werden, gewählt werden (für den &quot;trans&quot; Modell). Das Transposon Sleeping Beauty Systems erfolgreich der funktionellen Genomik, Insertionsmutagenese, Transgenese und somatische Gentherapie 21 verwendet werden. Als synthetisches Konstrukt zu einem funktionellen Molekül aus einem ruhenden Lachs Variante überarbeitet, wird die Dornröschen Transposase nicht auf andere Transposons in Menschen oder anderen Säugetieren 20 zu binden. Seit seiner Entdeckung hat Molekulartechnik enhanced die Umsetzung Wirksamkeit des SB Transposonsystem durch Veränderungen in der IR-Sequenzen und die Zugabe von TATA Dinukleotide flankierenden Transposon, was zu den pT2 Transposons. Diese Transposons haben an den IR-Website Bindung des SB optimiert und erhöht die Wirksamkeit der Exzision. Die SB Transposase unterzog auch signifikante Verbesserung; die Transposase in Experimenten hierin dargestellt verwendet wird, ist die SB100X, ein hyperaktives Transposase von einer DNA shuffling Strategie gefolgt von einer großen genetischen Screen in Säugetierzellen 22 erzeugt. In diesem Bericht zeigen wir eine schnelle, flexible und reproduzierbares Verfahren zur intrinsischen GBM in Mäusen, die mit nicht-viralen, Transposon-vermittelte Integration von Plasmid-DNA in Zellen der Unter ventrikulären Zone des neugeborenen Mäusen 23 induzieren. Wir präsentieren Ihnen eine einfache Möglichkeit, Transposons mit neuer Gene zugänglich für genomische Integration mit dem Dornröschen Transposase zu schaffen und zeigen, wie man das Plasmid Aufnahme zu überwachen undFortschreiten der Krankheit mit Biolumineszenz. Wir charakterisieren auch histologischen und immunhistochemischen Eigenschaften der GBM mit diesem Modell wiedergegeben. Darüber hinaus präsentieren wir Ihnen eine schnelle Methode, um primäre GBM Zelllinien aus diesen Tumoren zu erzeugen. Dornröschen Modell, bei denen Tumore aus Zellen Original dem Tier induziert werden, erlaubt das funktionale Bewertung der Rolle von Kandidaten GBM Gene bei der Induktion und Progression von Tumoren. Dieses System ist auch für das Testen von neuen GBM Behandlungen, einschließlich Immuntherapien in immunkompetenten Mäusen geeignet, ohne die Notwendigkeit invasiver entzündlichen chirurgischer Prozeduren, die die lokale Mikroumgebung verändern können.

<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.

Um histopathologischen Merkmale SB induzierten Glioblastome charakterisieren, C57 / BL6 neugeborenen Mäusen wurden an P1 mit einem Plasmid, das für Luciferase (PT2 / SB100x-Luc) in Kombination mit Plasmiden, Transposons Codieren mit onkogener DNA, dh NRAS (pT / CAGGS spritzt -NRASV12) und SV40 LgT (Pt / CMVSV40-LgT) (3c) oder ein Plasmid, das eine kurze Haarnadel-p53 mit PDGFβ und GFP-Reporter (pT2shp53 / GFP4 / mPDGFβ) in Kombination mit NRAS (Abbildung 3d Codierung). Die ...

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.

Transposon-vermittelte Integration von Plasmid-DNA in die Subventrikularzone von neugeborenen Mäusen zu Novel Modelle von Glioblastoma generieren

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 Aufrufe.  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.

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

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.

Die Verwendung Tiermodell für Sepsis zu neuartigen Therapien zu beurteilen Herbal

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

Forschung

Medizin

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

Identifizierung von<em&gt; Dornröschen</em&gt; Transposon Insertionen in Solid Tumors mit Linker-vermittelte PCR

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.

Bildgestützte Konvektion-enhanced Lieferung in Agarose-Gel-Modelle des Gehirns

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.

Orthotopische Injektion von Brustkrebszellen in das Brustfettpolster von Mäusen, das Tumorwachstum zu studieren.

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.

Maus Nierentransplantation: Modelle der Transplantatabstoßung

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.

Ein Ferkel Modell Neonatal hypoxischischämischer Enzephalopathie

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.

Chirurgische Modelle aus gastroösophagealen Reflux mit Mäusen

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.

Myokardinfarkts bei neugeborenen Mäusen, ein Modell von 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 ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

In Vivo Photolabeling von Zellen im Darm wandernden Potential der hämatopoetischen Zellen bei Neugeborenen Mäusen zu bewerten

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Full-Endoscopic Isolation Zone Technique for the Treatment of Lumbar Disc Herniation

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its clinical symptoms often include severe low back pain, limited lumbar movement, sciatic nerve pain in the lower limbs, and even cauda equina syndrome. The common treatment for LDH is a conservative treatment scheme involving medicine, rest, and physical therapy. However, if the conservative treatment scheme is ineffective, a surgical treatment approach is adopted. Traditional open lumbar surgery has some disadvantages, including the potential for severe surgical trauma, severe blood loss during the operation, instability of the lumbar spine, and loss of the lumbar motor unit. Among the minimally invasive surgical schemes, full-endoscopic spine surgery (FESS) is undoubtedly the most appropriate and has the advantages of minimal trauma, high safety, quick postoperative recovery, and the retention of the stable structure and the motor unit of the lumbar spine. However, simultaneously, incomplete removal of the nucleus pulposus and residual nerve dysfunction after surgery can occur. To avoid these shortcomings, we studied a specific spinal endoscopy technique, the "isolation zone" surgical strategy, which can effectively block the pain from the nerve conduction pathway by completely relieving the nerve compression and nerve dysfunction through the orderly treatment of the protruding nucleus pulposus, the fissure of the annulus fibrosus, the sinus nerve, and the surrounding inflammatory soft tissues.

Here, we present a protocol for the &#34;isolation zone&#34; technique in the treatment of lumbar disc herniation (LDH) under full-endoscopic spine surgery (FESS), including intervertebral foramen formation, targeted catheterization, nucleus pulposus resection, and annulus fibrosus formation, which together completely block the pain from the nerve conduction pathway.

Vollendoskopische Isolationszonentechnik zur Behandlung von lumbalen Bandscheibenvorfällen

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its ...