This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).

Questo studio è stato approvato e realizzato in conformità con le raccomandazioni del Comitato Istituzionale Animal cura e l&#39;uso presso l&#39;Università della California, Irvine. Tutte le procedure sono state eseguite in anestesia isoflurano e tutti gli sforzi sono stati fatti per ridurre al minimo le sofferenze. 1. Generazione di Ecotropic Retrovirus <ol><li> Preparare plasmidi di alta qualità, con una concentrazione di almeno 1 mg / mL utilizzando un kit di maxi-prep commerciale o purificazione di cloruro di cesio. NOTA: Queste includono un vettore backbone MSCV codificante il gene di interesse (in questo caso CALR 7,8) e marker di scelta (GFP, Neo, ecc) così come un plasmide di confezionamento ecotropic, codifica gag-pol-env (denominato qui come plasmide). </li><li> Scongelare ed espandere cellule 293T bassa di passaggio in DMEM supplementato con 10% FBS e L-glutammina con la penicillina / streptomicina (D10). Piastra 5 x 10 6 cellule in un 10 centimetri di tessuto piatto di coltura trattata in un umidificatacoltura tissutale incubatore a 37 ° C e 5% CO 2. </li><li> Espandere le cellule 293T in coltura. Il giorno prima trasfezione, lavare le cellule con l&#39;aggiunta di 1 ml di PBS e aspirare il surnatante in un pallone a vuoto. </li><li> Per trypsinize cellule, aggiungere 2 ml 0,05% tripsina e incubare piatti in umidificato coltura tissutale incubatore per 2 - 3 min a 37 ° C e 5% CO 2. </li><li> Per interrompere tripsinizzazione, aggiungere 3 ml D10 a piatto e le cellule di trasferimento ad un tubo da 15 ml. Spin cellule a 400 xg per 10 min. </li><li> Aspirare il surnatante in una boccetta di vuoto e risospendere pellet di cellule in 2 ml D10. </li><li> Contare le cellule di esclusione trypan blu utilizzando un emocitometro. </li><li> Seed 2 x 10 6 cellule per piatto in tre 10 cm di tessuto piatti della cultura trattati per virus generati. NOTA: Per ottenere risultati ottimali, non consentono alle cellule di superano il 30 - 50% confluenza al momento della trasfezione o di una cattiva resa virus può essere ottenuto. </li><li> Immediatamente prima trasfezione,mezzi aspirato in un pallone a vuoto e sostituirli con 5 ml D10 fresco. </li><li> Per ogni 10 cm Petri da transfettate preparare un individuo sterile provetta da 1,5 ml microcentrifuga. </li><li> Pipettare 760 ml di mezzi siero ridotto in ciascuna provetta, quindi aggiungere con cautela 40 ml di reagente di trasfezione direttamente nel supporto. Incubare le provette nella cappa coltura tissutale a temperatura ambiente per 5 min. </li><li> Aggiungere 30 mg di MSCV vettore con il gene di interesse (15 mcg se si utilizza vettore vuoto) e 5 mg di plasmide per ogni rispettivo tubo e mescolare delicatamente pipettando su e giù. Incubare a temperatura ambiente nella cappa coltura tissutale per altri 15 min. NOTA: i vettori più piccoli tendono ad avere rendimenti più elevati virali. </li><li> Aggiungere con cautela il volume totale di ogni reazione (circa 800 ml) goccia a goccia nel rispettivo 10 centimetri Petri e inclinare leggermente il piatto da un lato all&#39;altro per mescolare. Rientro cellule per il cu tessuti umidificataincubatore lture. </li><li> A seguito di una 8 ore o l&#39;incubazione durante la notte in coltura tissutale incubatore, i media aspirato in una boccetta di vuoto e delicatamente sostituire con 10 ml di D10 fresco. </li><li> A 48 ore dopo la trasfezione raccogliere surnatante in una siringa da 35 ml e filtrare attraverso una membrana da 0,45 micron per rimuovere i detriti cellulari. </li><li> OPTIONAL: 10 ml D10 fresca torna sulle cellule 293T e virus supplementare può essere raccolto in 72 ore dopo la trasfezione. </li><li> Smaltire cellule 293T in rifiuti biologici in assenza di virus vengono raccolte. </li><li> surnatante virale Aliquota in volumi monouso (ad esempio 1 ml per titolo virale o 6.5 ml per l&#39;infezione da BM). virus Flash-congelamento con azoto liquido e virus conservare a -80 ° C. NOTA: Evitare ripetuti cicli di congelamento / scongelamento in quanto riducono notevolmente titolo virale. </li></ol> 2. Determinare relativo titolo virale mediante citometria di flusso <ol><li> Scongelare 3T3 e mantenere in D10. Coltivare le cellule in un cm 10tessuto-coltura trattata piatto in un incubatore umidificato coltura tissutale a 37 ° C e 5% CO 2. </li><li> Eseguire titolo virale in triplice copia. Per preparare le cellule, lavare le cellule con l&#39;aggiunta di 1 ml di PBS e aspirare il surnatante in un pallone a vuoto. </li><li> Per trypsinize cellule, aggiungere 2 ml 0,05% tripsina e incubare piatti in un incubatore umidificato coltura tissutale a 37 ° C e 5% CO 2. </li><li> Per interrompere tripsinizzazione, aggiungere 3 ml D10 a piatto e le cellule di trasferimento ad un tubo da 15 ml. Spin cellule a 400 xg per 10 min. </li><li> Aspirare il surnatante in una boccetta di vuoto e risospendere pellet di cellule in 2 ml D10. </li><li> Contare le cellule di esclusione trypan blu utilizzando un emocitometro. </li><li> Seme 3T3 con una densità di 1 x 10 5 cellule in quindici tessuti piatti 60 mm coltura trattata e incubare durante la notte. </li><li> Rendere 1: 3, 1:10, 1:30 e 1: 100 diluizioni del surnatante virale scongelato utilizzando D10 in un totale di 1 ml. Anche un controllo privo di virus. </li><li> mi aspiraredia da 3T3 in una beuta da vuoto e sostituirlo con 4 ml di D10. </li><li> Sovrapponete le cellule 3T3 con le diluizioni di surnatante virale e aggiungere polibrene ad una concentrazione finale di 8 mg / ml per ogni piatto. Incubare per 48 ore in coltura tissutale incubatore a 37 ° C e 5% CO 2. </li><li> Determinare titolo virale relativa mediante citometria di flusso: <ol><li> Per lavare le cellule, aspirare i media in un pallone vuoto e aggiungere 5 ml di PBS. </li><li> Aspirare il PBS in un pallone a vuoto. Aggiungere 1 ml di 0,05% tripsina e incubare a 37 ° C per 2 - 5 minuti per staccare le cellule dal piatto. </li><li> Aggiungere 3 ml D10 e pipetta su e giù per staccare i restanti cellule dalla piastra. Trasferire le cellule in una provetta FACS e centrifugare a 400 xg per 10 min. </li><li> Decantare il surnatante e lavare le cellule con 3 ml di PBS. Centrifugare a 400 xg per 10 minuti e il surnatante decantare. Risospendere in 200 l di PBS. </li><li> Cellule eseguito su un citofluorimetro 9. Raccogliere tra 20.000 - 30.000 eventi all&#39;interno tegli vive porta cellulare (determinata dalla FSC e SSC) e calcolare la percentuale di GFP + e GFP - cellule. NOTA: Il titolo virale è considerato accettabile 1:30 diluizione virale rendimenti surnatante almeno 50% GFP + cellule 3T3. Se poi i risultati non ottimali di trasduzione possono ottenere questo risultato diluizione nelle cellule meno del 50% GFP +. Il calcolo per determinare titolo virale è stato adattato da Limon et al. , Sangue (1997) 9. </li><li> Per determinare relativo titolo del virus, utilizzare l&#39;equazione: titolo del virus = unità formanti di particelle (PFU) / ml = [(Numero di 3T3 × Frequenza di GFP + cellule) / volume del surnatante (ml)] × Fattore di diluizione. </li></ol></li></ol> 3. trasdurre donatori cellule del midollo osseo <ol><li> Per preparare topi donatori per il midollo osseo (BM) raccolto, prima anestetizzare topi di vaporizzatore isoflurano con una portata di 5% supplementare combinatoossigeno a 1 L / min. Pinch piede per valutare la reattività. NOTA: i topi donatori (meno di 8 settimane di età) devono essere preparati 5 giorni prima della raccolta di midollo osseo (8 giorni prima del trapianto). Uno topi donatori forniranno abbastanza cellule per almeno 2 destinatari. </li><li> Iniettare 150 mg / kg 5-fluorouracile (5-FU) tramite iniezione retro-orbitale nei topi anestetizzati con un 27 G × ½ &quot;ago. ATTENZIONE! 5-FU un agente chemioterapico antitumorale. Maneggiare sempre 5-FU all&#39;interno di una cappa certificata o un armadio biosicurezza canalizzata. Consultare il comitato di sicurezza di laboratorio e degli animali dell&#39;istituzione per determinare un&#39;adeguata etichettatura dei topi trattati con 5-FU. <ol><li> Posizionare il mouse anestetizzato su un lato e frenare utilizzando sia il pollice e l&#39;indice tale che l&#39;occhio sporge leggermente dalla testa. </li><li> Partendo laterale ago al canto mediale e con lo smusso rivolto verso l&#39;alto, inserire l&#39;ago con un 45° l&#39;angolo in direzione mediale e fermarsi quando è a metà strada inserito l&#39;ago. Lentamente iniettare le cellule e rimuovere rimuovere con attenzione l&#39;ago. NOTA: Se questa tecnica viene eseguita correttamente, l&#39;occhio deve rientrare un po &#39;e nessuna resistenza deve essere sentito al momento dell&#39;inserimento dell&#39;ago. </li><li> Esaminare il mouse per eventuali segni di lesioni, come gonfiore o sanguinamento. </li></ol></li><li> Harvest midollo osseo 5 giorni dopo il 5-FU trattamento (3 giorni prima del trapianto). <ol><li> Anestetizzare topi di vaporizzatore isoflurano con una portata di 5% ossigeno supplementare combinata 1 L / min. Pinch piede per valutare la reattività. </li><li> Sacrifice mouse dislocazione cervicale. Sterilizzare il mouse a spruzzo generosamente con il 70% EtOH fino pelliccia si bagna. </li><li> Utilizzando dissezione forbici, fare una incisione nella pelle e sezionare la fascia lontano dai muscoli delle gambe. Successivamente, sezionare la gamba dal mouse recidere il femore a livello dell&#39;anca e la tibia alla caviglia. </li><li> piegarela gamba in una forma a &quot;L&quot; e separare ogni osso della gamba tagliando al ginocchio con le forbici dissezione. </li><li> Per rimuovere la cartilagine alle estremità distali di ciascun osso della gamba, utilizzare le forbici dissezione per raschiare delicatamente muscolare verso una estremità della gamba ossa fino cartilagine è disimpegnato. </li><li> Usando una siringa con un ago 27 G x ½ pollici, flush ogni osso gamba con PBS integrato con il 2% FBS su una membrana di nylon 100 mM in una provetta conica da 50 ml. osso a filo fino a quando l&#39;osso diventa bianco per massimizzare il numero di cellule raccolte. </li><li> Utilizzare lo stantuffo di una siringa per schiacciare cellule attraverso la membrana di nylon nel tubo conico 50 ml per ottenere una sospensione di cellule singole. Ripetere con l&#39;altra gamba del mouse. </li></ol></li><li> cellule centrifugare a 4 ° C per 10 min a 400 xg ed eliminare il surnatante. Risospendere BM in 5 ml di cloruro di ammonio di potassio (ACK) buffer per lisare i globuli rossi. Incubare le cellule in ghiaccio per 10 min. </li><li> Riempire il tubo con PBS per fermare la lisi cellulare. Centricellule Fuge a 4 ° C per 10 min a 400 xg ed eliminare il surnatante. Ripetere il passaggio lisi se il pellet BM ha colore rosso. </li><li> Risospendere in 5 ml di PBS e contare le cellule di esclusione trypan blu utilizzando un emocitometro. </li><li> Risospendere le cellule a 2 x 10 6 cellule per m con 2x cocktail pre-stimolazione contenente DMEM integrato con 20% FBS, L-glutammina, penicillina / streptomicina, IL-3 (14 ng / ml), IL-6 (24 ng / ml) e SCF (112 ng / ml). Piastra 2 ml per pozzetto in 6 pozzetti. </li><li> Incubare le cellule per una notte a 37 ° C e 5% CO 2. </li><li> Aggiungere 2 ml di surnatante virale ad ogni pozzetto contenenti cellule BM e aggiungere polibrene (8 mg / ml). piastre Spin a 30 ° C per 90 min a 1500 xg per poi tornare piastra per coltura tissutale incubatore overnight. </li><li> sospensione cellulare Pipettare in tubi conici e centrifugare a 4 ° C per 10 min a 400 xg, non gettare la piastra. Risospendere le cellule in 2 ml fresca 2x cocktail pre-Stim per bene e tornare ai pozzi originali. </li&gt; <li> Eseguire un spinoculation 2 ° ripetendo i passaggi 3.7-3.9. </li></ol> 4. Cellule trapianti in topi riceventi <ol><li> Preparare topi riceventi singenici per il trapianto con dose letale irradiazione corporea totale &lt;24 ore prima dell&#39;iniezione di cellule BM. NOTA: irradiazione dose letale per / c topi BALB è tipicamente 800-900 cGy e 1000-1200 cGy per C57B / 6 10. La dose è di solito divisa in due sessioni di irradiazione distanziati di almeno 3-4 ore di distanza. </li><li> Continua da punto 3.11, le cellule del donatore raccolto trasdotte dai 6 pozzetti pipettando delicatamente surnatante in tubi conici. Lavare ogni pozzetto con 1 ml di PBS per raccogliere le cellule residue e aggiungere a 50 ml provette coniche. </li><li> Aggiungere 0,5 ml di 0,05% tripsina ai pozzetti e incubare a 37 ° C in incubatore cultura tissutale per 5 min. </li><li> Aggiungere 1 ml D10 di pozzi e staccare eventuali cellule rimanenti delicatamente pipettando. Aggiungere cellule al rispettivo tubos dal punto 4.2 e pellet per centrifugazione a 4 ° C per 10 min a 400 x g. </li><li> Lavare le cellule con PBS e centrifugare a 4 ° C per 10 min a 400 x g. Risospendere in 5 ml di PBS e contare le cellule utilizzando un emocitometro da trypan blu. NOTA: Se il vettore ha un tag GFP quindi di quantificare la percentuale di cellule GFP-positive con un citofluorimetro. </li><li> Risospendere 5 x 10 5 - 2 x 10 6 cellule in 50 - 100 microlitri di PBS per iniezione in topi. </li><li> Iniettare cellule tramite iniezione retro-orbitale in topi anestetizzati con una sterile 27 G x ½ &quot;ago. NOTA: Altre tecniche di iniezione includono coda vena splenica o femorale. <ol><li> Anestetizzare il mouse con isoflurano e un pizzico piedi per valutare la reattività. </li><li> Posizionare il mouse su un lato e frenare utilizzando sia il pollice e l&#39;indice tale che l&#39;occhio sporge leggermente dalla testa. </li><li> Con lo smusso rivolto verso l&#39;alto, inserire il nEedle lateralmente al canto mediale a 45 °. NOTA: Se questa tecnica viene eseguita correttamente, l&#39;occhio deve rientrare un po &#39;e nessuna resistenza deve essere sentito al momento dell&#39;inserimento dell&#39;ago. </li><li> Esaminare il mouse per eventuali segni di lesioni, come gonfiore o sanguinamento. NOTA: i topi trapianto seguito dovranno emocromo bassi per un certo numero di settimane e quindi sono suscettibili alle infezioni. Profilassi con antibiotici o uso di acqua acidificata può essere utilizzato. </li></ol></li><li> Un mese dopo il trapianto, misurare la GFP% nel sangue periferico come un indicatore per le cellule del donatore trapiantate mediante citometria di flusso. NOTA: GFP da sola non può determinare il successo di attecchimento delle cellule. La trasduzione virale potrebbe non essere riuscito ancora attecchimento potrebbe essersi verificato. In questo caso, non le cellule GFP + sarebbero rilevati, ma l&#39;attecchimento delle cellule trapiantate ha avuto successo. <ol><li> Prendere 10 ml di sangue periferico By la puntura della vena safena della gamba con un 21 G x 1 ½ &quot;siringa e aggiungerlo a 100 mm EDTA in PBS per prevenire la coagulazione. </li><li> Aggiungere 1 ml di ACK al sangue e lisare in ghiaccio per 10 min. Cellule pellet per centrifugazione a 400 xg per 10 min. </li><li> Aspirare il surnatante in un pallone vuoto e aggiungere 1 ml di PBS integrato con il 2% FBS per fermare lisi. Cellule pellet per centrifugazione a 400 xg per 10 min. </li><li> Aspirare il surnatante in un pallone vuoto e risospendere pellet in 100 ml di PBS integrato con il 2% FBS. </li><li> Esegui campioni su un citofluorimetro e raccogliere 300.000 eventi all&#39;interno del cancello cellule vive (determinata dalla FSC e SSC) e calcolare la percentuale di GFP + e GFP - cellule 9. </li></ol></li><li> Inoltre a 1 mese post-trapianto, effettuare emocromo completo (CBC) utilizzando un processo automatizzato analizzatore ematologico veterinaria 18 per monitorare la progressione della malattia. NOTA: Continuare a monitorare la malattia da%GFP nel sangue periferico e CBC mensile. </li><li> Per terminare l&#39;esperimento, anestetizzare il mouse con isoflurano e un pizzico piedi per valutare la reattività. Sacrifice mouse dislocazione cervicale e la raccolta della milza e del midollo osseo per istopatologia. NOTA: La durata dell&#39;esperimento è personale e la data di scadenza dovrebbe essere valutata sulla base della malattia fenotipo atteso. </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+myeloproliferative+neoplasms:+JAK+of+all+grades.">Mouse models of myeloproliferative neoplasms: JAK of all grades.</a> Dis. Model. Mech. 4 (3), 311-317 (2011).</li><li>Mullally, A., Lane, S. W., Brumme, K., Ebert, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloproliferative+neoplasm+animal+models.">Myeloproliferative neoplasm animal models.</a> Hematol. Oncol. Clin. North Am. 26 (5), 1065-1081 (2012).</li></ol>

The authors have nothing to disclose.

Questo protocollo fornisce una descrizione dettagliata di come eseguire trapianti di midollo osseo in topi ricapitolare una malattia essenziale trombocitemia simile con la progressione di mielofibrosi con CALRdel52 mutazione come il conducente della malattia. il trapianto di successo di cellule che esprimono i risultati CALRdel52 un aumento delle piastrine, l&#39;espansione dei megacariociti, e fibrosi del midollo osseo. Come trapianto di midollo osseo è un processo a più fasi, è importante riconoscere passaggi dove ...

Trasduzione-trapianto è un metodo utile per modellare neoplasie ematologiche nei topi. Questa tecnica è stata particolarmente utile per lo studio di neoplasie mieloidi risalenti alla prima dimostrazione che l&#39;espressione ectopica di BCR-ABL1 potrebbe fedelmente ricapitolare leucemia mieloide cronica nei topi 1. Questa tecnica ha successivamente facilitato l&#39;ampio studio di JAK2 V617F e MPL W515K / L mutato neoplasia mieloproliferativa (MPN). MPN sono un gruppo di neoplasie ematologiche caratterizzate dalla sovrapproduzione di cellule mieloidi mature e fibrosi del midollo osseo. Queste malattie in genere derivano dall&#39;espansione clonale di una cellula staminale emopoietica che ha acquisito una mutazione somatica in entrambe le Jak2, MPL, o CALR. Trasduzione-trapianto di JAK2 V617F e MPL W515K / modelli L mostra le caratteristiche cliniche della policitemia vera e mielofibrosi 2-5 &lt;/ Sup&gt;. Di recente, un modello murino di calreticulin-mutato MPN è stato anche generato con il metodo di trasduzione-trapianto 6. Questi topi sviluppano una malattia trombocitemia essenziale simile ad un aumento delle piastrine, aumento del numero di megacariociti, e fibrosi del midollo osseo. Insieme, questi modelli non solo hanno fornito l&#39;occasione al fine di conoscere la patogenesi molecolare di MPN, ma anche la capacità di sviluppare e studiare terapie in un ambiente pre-clinica. Questo manoscritto fornisce una descrizione dettagliata della metodologia di trasduzione-trapianto con una particolare attenzione per la mutazione CALRdel52. Questa tecnica prevede il trapianto di cellule del midollo osseo retrovirally trasdotte che esprimono il mutante costrutto in topi riceventi singenici irradiati.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>MT-10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td></td>
		</tr>
		<tr>
			<td>PLASMIDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EcoPak, also known as pCL-Eco</td>
			<td>Addgene</td>
			<td>12371</td>
			<td>Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional &#947;-retrovirus envelope and packaging plasmids are available from Addgene and others.</td>
		</tr>
		<tr>
			<td>MSCV-IRES-GFP (MIG)</td>
			<td>Addgene</td>
			<td>20672</td>
			<td>Additional &#947;-retroviral transfer plasmids are available from Addgene and others.</td>
		</tr>
		<tr>
			<td>CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G x 1/2&#34; needles</td>
			<td>BD</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>MT-35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/L-glutamine</td>
			<td>Corning</td>
			<td>MT-30-009-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Corning</td>
			<td>MT-25-052-CI</td>
			<td>Can be homemade</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>MT-21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm dishes</td>
			<td>Fisher</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Fisher</td>
			<td>12565268</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm dishes</td>
			<td>Fisher</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td></td>
		</tr>
		<tr>
			<td>5-FU</td>
			<td>Fisher</td>
			<td>A13456-06</td>
			<td></td>
		</tr>
		<tr>
			<td>100um cell strainers</td>
			<td>Fisher</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Fisher</td>
			<td>12565270</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Fisher</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>14-959-5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45um syringe filters</td>
			<td>Fisher</td>
			<td>0974061B</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Can be homemade</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-6</td>
			<td>Peprotech</td>
			<td>216-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365809001</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray irradiator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transduction-transplantation mouse model of myeloproliferative neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

La tecnica trasduzione-trapianto comporta ricostituzione ematopoietica dei topi riceventi con cellule esprimenti il ​​gene di interesse. La Figura 1 mostra una panoramica del modello di topo trasduzione-trapianto di calreticulin mutato MPN. In breve, retrovirus che esprimono CALRwt o CALRdel52 viene utilizzato per infettare le cellule BM da un topo donatore C57B / 6. cellule trasdotte vengono trapiantate in irradiato C57B / 6 del mouse ricevente e donatore di attecch...

Watch this Scientific Journal Video about Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm at JoVE.com

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Trasduzione-trapianto modello murino di neoplasia mieloproliferativa

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

transduction-transplantation-mouse-model-myeloproliferative

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JoVE Journal

Cancer Research

11.8K Views.  University of California, Irvine. This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation. 

Video: Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluorescente ortotopico modello murino di cancro al pancreas

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

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Ricerca sul cancro

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Un modello murino di fatica indotta da irradiazione Peripheral

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Isolamento delle cellule tumorali circolanti in un modello di topo ortotopico del tumore del colon-retto

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Un modello di mouse geneticamente modificato del cancro colorettale sporadico

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

Attecchimento del tumore in un modello murino di xenotrapianto di umano linfoma a cellule mantellari

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Un modello murino di cancro del pancreas Syngeneic per studiare gli effetti dell'elettroporazione irreversibile

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Impianto ortotopico a ultrasuoni dell'adenocarcinoma dugalese Murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Un modello ortotopico di topo resezionale del cancro al pancreas

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Tecniche molecolari e immunologiche in un modello murino geneticamente modificato di tumore stromale gastrointestinale

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Ex Vivo Modello organoide delle delezioni geniche mediate da Adenovirus-Cre in cellule uroteliali di topo

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...