We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

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The authors have nothing to disclose.

Con l&#39;automobile e HSC-based immunità ingegnerizzato acquistando slancio verso gli studi clinici, è importante avere un modello animale adeguato per esaminare da vicino la differenziazione e la funzione di queste cellule ingegnerizzate. In questo protocollo abbiamo descritto i metodi per costruire e testare i topi umanizzati con cellule staminali geneticamente modificate ingegnerizzate contro l&#39;HIV. E &#39;importante avere efficiente trasduzione di cellule staminali prima del trapianto. Tuttavia, a causa della capacità di cellule T a proliferare previo riconoscimento delle cellule bersaglio, bassi livelli di modificazione di cellule staminali sono sufficienti a generare una risposta forte contro la replicazione dell&#39;HIV 8. Tuttavia, per raggiungere alto livello di modificazione delle cellule staminali, si consiglia di utilizzare CD34 + trasdotte cellule in miscela proteica gelatinosa con timo autologo, invece di fegato e timo pezzi per la chirurgia topi che erano stati descritti altrove 13-15. miscela proteica gelatinosa è un solubiliztessuti ed membrana basale ricco di proteine ​​della matrice extracellulare. In condizioni fisiologiche normali, miscela proteica gelatinosa polimerizzare per produrre una matrice ricostituito, biologicamente attivo e stabile che consenta fissaggio efficace e la differenziazione delle cellule staminali 20. la ricostituzione delle cellule umane può essere controllato da emorragia retro-orbitale e citometria a flusso 6 - 8 settimane dopo l&#39;intervento chirurgico. Per garantire che il vettore non è tossico per la sopravvivenza delle cellule staminali e rinnovo, si raccomanda di poi titolare il vettore in cellule CD34 + prima dell&#39;esperimento come descritto in 1.2.6. La capacità del modello di topo NSG-BLT per supportare l&#39;infezione della mucosa, viremia coerente e risposte immunitarie cellulari lo rende un modello molto utile per studiare l&#39;HIV patologia immunitaria e le terapie a base di cellule per il trattamento dell&#39;infezione da HIV 1. Ancora più importante, le cellule T generate dai topi NSG-BLT sono selezionati nel tessuto del timo autologo, consentendo al ricercatore di studiare il destinodi cellule staminali gene modificato dopo la selezione del timo 8,21. Con il metodo descritto, siamo stati in grado di ottenere il 40% -90% la ricostituzione delle cellule immunitarie umane in modo coerente. Bassi livelli di ricostituzione cellulare umana possono derivare da molteplici fattori, tra cui le competenze della persona che effettua l&#39;intervento chirurgico e la qualità dei tessuti per il trapianto. Per ottenere un&#39;elevata ricostituzione cellula umana, è importante garantire i trapianti siano posizionate saldamente sotto la capsula renale. Inoltre, si raccomanda di esaminare ogni impianto timica preparato per un intervento chirurgico sotto microscopio ottico ed eliminare eventuali pezzi discutibili. Anche se il modello umanizzato BLT mouse è uno strumento promettente per studiare l&#39;immunità ingegnerizzato contro l&#39;HIV (rivisto in 1,15,22), ha i suoi limiti. Vale a dire, questo modello non perfettamente imitare un sistema immunitario periferico umano. Gli studi hanno dimostrato lo sviluppo alterato del, Antibo IgG di classe a commutazione di iper-mutatody 1,23. Inoltre, utilizzando topi immuno-deficienti è tecnicamente impegnativo e mantenere questi topi sani richiede notevoli risorse e formazione. infezioni opportunistiche sottili possono manifestarsi differenze significative tra i campioni e potenzialmente avere conseguenze negative sulla sperimentazione. Pertanto, è importante avere strutture ben preparati e personale adeguatamente formato per mantenere l&#39;integrità del futuro 1,24 dati. Con queste limitazioni in mente, il modello di topo NSG-BLT umanizzato fornisce ancora un importante strumento per lo studio della immunità ingegnerizzato basato sulle cellule staminali, come dimostrano questi esempi 4,8. Con la tendenza di sviluppare recettori chimerici antigene basato su HIV ampia anticorpi neutralizzanti 10 e la modifica del dominio di segnalazione per più efficiente CAR 25, questo modello e protocollo possono essere utilizzati per caratterizzare e studiare la funzionalità del gene modificato celLS con una nuova generazione di automobili. Inoltre, questo modello può potenzialmente ospitare studi sulla terapia a base immunitaria (come inibitorio blocco dei recettori), in combinato disposto con l&#39;immunità Engineered.

Nonostante il successo del combinato terapia anti-retrovirale, l&#39;infezione da HIV è ancora una malattia per tutta la vita. La risposta immunitaria cellulare contro l&#39;HIV gioca un ruolo molto importante nel controllo della replicazione di HIV. I recenti progressi nella manipolazione di cellule staminali ha permesso per il rapido sviluppo di approcci di terapia genica per l&#39;HIV trattamento 1-3. Di conseguenza, è importante avere un modello animale adeguato che permette studio in vivo dell&#39;efficacia di terapie cellulari contro l&#39;HIV. Utilizzo di HIV in modelli animali è complicata dal fatto che il virus infetta soltanto cellule umane. Per aggirare questa limitazione, gli scienziati hanno fatto ricorso all&#39;utilizzo di modelli di malattia come il virus dell&#39;immunodeficienza delle scimmie (SIV) in Rhesus macachi 4,5. Purtroppo, ci sono importanti limitazioni in questo modello a causa delle differenze intrinseche tra le specie e le differenze tra SIV e HIV. Inoltre, solo i servizi altamente specializzati sono capable di sostenere il lavoro con i primati non umani e ogni macaco richiede un grande investimento. Pertanto, vi è una necessità urgente di un modello che utilizza il sistema immunitario umano, che è suscettibile di infezione HIV / patogenesi, ed è meno proibitivo finanziariamente. Il diabetici non obesi (NOD) -Grave combinato immunodeficienti (SCID) -Comune gamma catena knockout (c γ - / -) (o NSG) Sangue / Fegato / Thymus (BLT) modello di topo umanizzato è sempre dimostrato di essere uno strumento importante per studiare l&#39;infezione da HIV. Impiantando cellule staminali ematopoietiche (CSE) e del timo fetale, i topi sono in grado di sviluppare e riepilogare un sistema immunitario umano 1-3. Un tipo di terapia genica basata sulle cellule staminali coinvolge &#39;reindirizzamento&#39; cellule T periferiche di indirizzare l&#39;HIV riprogrammando cellule staminali ematopoietiche (CSE) di differenziarsi in cellule T antigene specifico. Abbiamo dimostrato in precedenza che HSC ingegneria con un molecolare clonato anti-HIV specifica re delle cellule Trecettore (TCR) contro l&#39;epitopo SL9 (aminoacido 77-85; SLYNTVATL) del virus HIV-1 gag può reindirizzare le cellule staminali in formazione di linfociti T maturi che sopprimono la replicazione di HIV nel umanizzato NSG-BLT modello di topo 6. L&#39;avvertimento di utilizzare un TCR clonato molecolare è che è limitato ad uno specifico sottotipo antigene leucocitario umano (HLA) che limitare l&#39;applicazione di questa terapia. recettori chimerici antigene (CAR), d&#39;altro canto, possono essere universalmente applicati a tutti i sottotipi HLA. Gli studi iniziali sono stati eseguiti utilizzando un CAR costruito con i domini extracellulari e transmembrana di CD4 umana fusa alla ζ segnalazione intracellulare dominio di CD3 (definito il CD4ζCAR). CD4ζCAR espresso sulle cellule T CD8 può riconoscere busta HIV e scatenare una risposta delle cellule T citotossiche che è simile a quella mediata da un recettore delle cellule T 7. Abbiamo recentemente dimostrato che cellule staminali emopoietiche umane possono essere modificati con CD4ζCAR, che possono poi differenziarsi in molteplici ematopoietiche Lineages, comprese le cellule T funzionali in grado di sopprimere la replicazione dell&#39;HIV nel modello umanizzato del mouse 8. Con il rapido avanzamento in chimerici terapie recettore antigene per il cancro 9, e la caratterizzazione continuo di potenti ampie anticorpi neutralizzanti 10-12 contro l&#39;HIV che permettono la costruzione di un&#39;unica catena CAR anticorpi, è percepibile che molti nuovi costrutti candidati, in aggiunta a CD4ζCAR , sarà generato e testato per la terapia genica a base di cellule staminali di malattie HIV e altre malattie. Inoltre, il modello di topo NSG-BLT umanizzato contenente queste vetture antigene-specifiche può anche fornire un utile strumento per esaminare attentamente le risposte delle cellule T umane in vivo. È importante sottolineare che il nostro protocollo differisce dai metodi descritti precedenti per la costruzione di umanizzato di topi BLT 13-15 dal fatto che la CSE in miscela proteica gelatinosa viene utilizzato al posto di tronchi fegato fetale 16. Questo protocollo descrive: 1) costruzione di Humanitopi BLT zed ingegnerizzati con CD4ζCAR; e 2) caratterizzazione del differenziamento delle cellule geneticamente modificate; e 3) caratterizzazione della funzionalità delle cellule geneticamente modificate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD34 microbead kit</td>
			<td>miltenyi</td>
			<td>130-046-702</td>
			<td>For sorting human CD34+ progenitor cells</td>
		</tr>
		<tr>
			<td>Bambanker</td>
			<td>Wako</td>
			<td>302-14681</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>For measuring viral load in the serum</td>
		</tr>
		<tr>
			<td>MACSQuant Flow Cytometer</td>
			<td>Miltenyi</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482;</td>
			<td>BD biosciences</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H6254-500MG&#160;</td>
			<td>For tissue digestion</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Worthington</td>
			<td>LS002006&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life technology</td>
			<td>17104-019&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>CFX Real time PCR detection system</td>
			<td>Biorad</td>
			<td></td>
			<td>For measuring viral load and gene expression</td>
		</tr>
		<tr>
			<td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td>For constructing the humanized mice</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10378016</td>
			<td>For culturing cells</td>
		</tr>
		<tr>
			<td>piperacillin/tazobactam</td>
			<td>Pfizer</td>
			<td>Zosyn</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>Amphotericin B (Fungizone antimycotic)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290-018</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm - 1000 units&#160;&#160;&#160;&#160;</td>
			<td>Becton Dickinson</td>
			<td>427631&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Medline</td>
			<td>DYNJP2707&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>sutures, 4-0, vicryl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>23000J304H&#160;&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Alcohol prep pads&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>3583006818</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Gloves, surgical, 6 1/2</td>
			<td>Owens and Minor</td>
			<td>4075711102</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Yssel&#8217;s Serum-Free T-Cell Medium</td>
			<td>Gemini Bio-products</td>
			<td>400-102</td>
			<td>For CD34+ cell transduction</td>
		</tr>
		<tr>
			<td>Human Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9511</td>
			<td>For CD34+ cell transduction</td>
		</tr>
	</tbody>
</table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

Con il rapido sviluppo di terapie geniche a base di cellule staminali contro l&#39;HIV, non vi è necessità di premere un modello animale per studiare la differenziazione ematopoietiche e la funzione immunitaria delle cellule geneticamente modificate. Il umanizzato midollo osseo / Fegato / Thymus (BLT) topo modello consente la piena ricostituzione di un sistema immunitario umano in periferia, che comprende le cellule T, cellule B, cellule NK e monociti. L&#39;impianto del timo umano consente anche per la selezione timica delle cellule T nel tessuto del timo autologo. Oltre allo studio dell&#39;infezione da HIV, il modello rappresenta un potente strumento per studiare la differenziazione, lo sviluppo e la funzionalità delle cellule derivate da cellule staminali ematopoietiche (CSE). Qui descriviamo la costruzione di umanizzato diabetici non obesi (NOD) -Grave combinato immunodeficienti (SCID) -Comune gamma catena knockout (c γ - / -) topo Hong osseo osseo / Fegato / Thymus (NSG-BLT) con CSE trasdotte con CD4 recettore per l&#39;antigene chimerico (CD4CAR)vettore lentivirus. Abbiamo dimostrato che il CD4CAR CSE può differenziarsi con successo in molteplici linee e hanno attività anti-HIV. L&#39;obiettivo dello studio è quello di dimostrare l&#39;uso del modello di topo NSG-BLT come un modello in vivo per l&#39;immunità ingegnerizzato contro l&#39;HIV. Vale la pena notare che, poiché viene utilizzato lentivirus e tessuti umani, esperimenti e interventi chirurgici devono essere eseguiti in una classe II armadio biosicurezza in un livello di biosicurezza 2 (BSL2) con particolari precauzioni (BSL2 +) struttura.

La figura 1 mostra uno schema di costruzione di topi BLT umanizzati con cellule staminali modificato. 10 settimane dopo la chirurgia implantare, i topi sono stati sacrificati per valutare la differenziazione e sviluppo di cellule geneticamente modificate. Come mostrato in figura 2, molteplici tessuti linfoidi (sangue, milza, timo e midollo osseo) sono state raccolte da un topo che è stato modificato con CD4ζCAR. Il CD4ζCAR utilizzato in questo protocollo contiene CD4 recettore per l&#39;antigene chimerico e GFP che può essere rilevato da anticorpi anti-CD4 e l&#39;espressione della GFP 8. Le cellule sono state isolate e colorati con anticorpo anti CD45 umana e anticorpo anti-CD4 e analizzate mediante citometria di flusso. GFP e CD4 doppie cellule positive sono state rilevate, indicando la presenza di cellule CD4CAR + in diversi tessuti linfoidi. Per studiare la differenziazione delle cellule geneticamente modificate,<html> splenociti sono state colorate con anticorpi contro CD45 umano (linfociti), CD3 (cellule T), CD19 (cellule B), CD14 (monociti e macrofagi) e CD337 (cellule NK). Come mostrato in figura 3, le cellule staminali ematopoietiche CD4ζCAR differenziarsi in molteplici linee. Per indagare se le cellule CD4CAR modificate sono funzionali, abbiamo coincubated splenociti con cellule bersaglio che le cellule CD4CAR sarebbero riconoscere (cellule infette da HIV T1 o cellule non infette T1 come controllo). Le cellule sono state co-incubate durante la notte e l&#39;inibitore della proteina di trasporto è stato aggiunto per ulteriori 6 ore. In seguito, le cellule sono state fissate e permeabilizzate a macchia per l&#39;espressione intracellulare di citochine come IFNΓ e TNF-alfa. Come mostrato in figura 4, le cellule che esprimono CAR prodotte maggiore quantità di IFNΓ e TNFa con cellule infette T1. carico / 54048 / 54048fig1.jpg &quot;/&gt; Figura 1: Schema di costruzione di topi BLT umanizzati con le cellule staminali modificate FT:. Timo fetale. FL:. Fegato fetale <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54048/54048fig2.jpg" /> Figura 2:. Cellule modificate CD4 recettore per l&#39;antigene chimerico possono essere rilevati in diversi tessuti linfoidi Mouse con CD4CAR modificato le CSE sono stati sacrificati 10 settimane dopo l&#39;intervento chirurgico e molteplici tessuti linfoidi sono state raccolte e le cellule sono state colorate con CD45 anti-uomo e anti-umane anticorpi CD4 e analizzate mediante citometria di flusso. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig2large.jpg" target="_blank">cliccate qui per vedere una versione più grande di questa figura.</a> Figura 3:.. Cellule modificate CD4 recettore per l&#39;antigene chimerico possono differenziarsi in molteplici linee splenociti di topi CD4CAR modificate sono state raccolte e colorati con anticorpi contro CD45 umano, CD3, CD19, CD14 e CD337 e analizzata mediante citometria di flusso <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig3large.jpg" target="_blank">Clicca qui per visualizzare un più grande versione di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54048/54048fig4.jpg" /> Figura 4:. Ex vivo saggio di citochine delle cellule T CD4 + CAR splenociti da HIV infetta i topi CD4CAR sono stati stimolati sia con cellule T1 infetti o non infetti da HIV e la loro produzione intracellulare di citochine è indicata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig4large.jpg" target="_blank">Cliccate qui per vie</a>wa più grande versione di questa figura.

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

Cellule staminali base Immunità Engineered contro l&#39;infezione da HIV nel modello umanizzato mouse

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

stem-cell-based-engineered-immunity-against-hiv-infection-humanized

Nanyang Technological University

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JoVE Journal

Medicine

10.1K Views.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

Video: Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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Medicina

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modello murino di Nell&#39;utero Trapianto

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Induzione farmacologica di melanina epidermica e di protezione contro le scottature in un modello di topo umanizzato

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation in Sperimentale Contusivi Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Una procedura chirurgica per la resezione Rib Mouse: Un modello per le grandi ossa lunghe Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Un neonatale mouse Spinal Cord Injury compressione Modello

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Vena interposizione Modello: un modello adatto per studiare Bypass Graft pervietà

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Basato su palloncino ferita per indurre iperplasia Myointimal nell'Aorta addominale di Mouse

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Cellule staminali base Immunità Engineered contro l'infezione da HIV nel modello umanizzato mouse

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