Nous remercions le Dr Charles Greenberg pour l&#39;utilisation de sa machine PCR en temps réel. Ce travail est soutenu par MUSC Hollings Cancer Center fonds de démarrage, Hollings Cancer Center ACS IRG, ASCO Conquer Cancer Foundation Award développement de carrière, le NIH 1K08HL 103780-01A1, et les NIH 3P30CA138313-01S3. Le contenu relève de la seule responsabilité des auteurs et ne reflètent pas nécessairement les vues officielles de la National Institutes of Health ou des agents de financement.

1. Isolement de cellules de moelle osseuse <ol><li> Euthanasier mâles donneurs FVB / NJ souris et femelles FVB / NJ souris utilisant le CO 2 méthode suivie par dislocation cervicale. Les femelles FVB / NJ cellules de moelle osseuse seront utilisées comme cellules compétitifs. </li><li> Utilisez de petits ciseaux et des pinces, disséquer les fémurs et tibiaes de souris et de les placer dans une boîte de culture tissulaire de 60 mm contenant 6 ml RPMI1640 glacée avec du FBS inactivé chaleur 5%. Utilisez le tissu Kimwipe pour enlever les muscles et d&#39;autres tissus. Couper les deux extrémités de chaque corps d&#39;os dans le plat. </li><li> Branchez l&#39;extrémité de l&#39;os avec une aiguille 23G sur une seringue de 3 cc, rincer la moelle osseuse avec RPMI1640 avec du FBS inactivé chaleur 5% dans le plat. Désagréger les tissus de moelle osseuse par des aspirations répétées en utilisant la même aiguille. Transférer la suspension cellulaire à 15 ml du tube de centrifugation. </li><li> Centrifuger les cellules pendant 5 min à 400 xg, éliminer le surnageant, remettre en suspension les cellules dans 1 ml de sang température ambiante tampon de lyse des globules rouges (155 mM de pbicarbonate de otassium, 10 mM de chlorure d&#39;ammonium, 0,1 mM d&#39;EDTA, pH = 7,4) et incuber à température ambiante pendant 5 min, puis ajoutez 5-10 ml de RPMI 1640 avec du FBS inactivé chaleur 5%. </li><li> Passez les cellules à travers un tamis cellulaire. Recueillir le débit à travers un nouveau tube. Centrifuger pendant 5 min à 400 x g. Retirer le surnageant, le culot cellulaire ne doit contenir aucune couleur rouge. L&#39;absence de couleur rouge indique une suppression complète des globules rouges. Remettre en suspension le culot cellulaire dans 10 ml de RPMI 1640 avec du FBS inactivé chaleur 5%. Vortexer doucement pour s&#39;assurer que la suspension cellulaire est complètement uniforme. Prélever une partie aliquote et compter les cellules dans un hémocytomètre. Calculez combien de volume de cellules nécessaires pour la transplantation de moelle osseuse et de cellules aliquotes et mélanger suffisamment de cellules du donneur avec des cellules mâles concurrents femelles à un ratio de 5:2. Centrifuger pendant 5 min à 400 xg, laver avec du PBS, remettre en suspension dans du PBS à la concentration finale de cellules du donneur à 5 x 10 cellules 6 / ml et un concurrent à 2 × 10 6 </sup&gt; / ml. </li></ol> 2. Concurrentiel greffe de moelle osseuse <ol><li> Souris irradier bénéficiaires féminins (8-12 semaines d&#39;âge) avec des rayons gamma radiateur à une dose unique de 11Gy 4-6 heures avant la transplantation de moelle osseuse 137Cs. </li><li> Placez les souris irradiées de sexe féminin dans un dispositif de retenue de la souris. Injecter le donateur mixte et les cellules concurrents via la veine caudale dans 0,1 ml de volume total de sorte que chaque souris reçoit 5 × 10 5 cellules du donneur et 2 × 10 5 cellules de la moelle osseuse concurrents. </li></ol> 3. Prélèvement des échantillons Couples semaine après la transplantation de moelle osseuse, de prélever des échantillons de sang périphérique (~ 50 pi) de la souris femelle receveuse par rétro-orbitaire saignement sous condition de l&#39;anesthésie. Les échantillons sont prélevés dans des tubes EDTA enduits. Échantillons BM peuvent également être recueillies à la fin de l&#39;expérience, le plus souvent au moins 4 post-transplantation mois, avec les mêmes procédures que décrites précédente (étape 1);% habituellement 20-40 du1 tibia cellules BM sont assez pour faire une quantité suffisante d&#39;ADN. Échantillons de sang ou de BM âge appariés normaux chez les souris femelles et mâles sont également recueillies pour préparer la courbe standard. 4. Isolement de l&#39;ADN génomique <ol><li> Ajouter 4 × volume de la chambre de lyse RBC température tampon (~ 200 pi) pour chaque échantillon de sang. Bien mélanger et incuber à température ambiante pendant 5 min. Ajouter 1 ml de PBS, puis tourner vers le bas pour enlever la plupart des lysé RBC. </li><li> Isoler l&#39;ADN en utilisant un kit d&#39;extraction de sang ADN (ADN QIAmp sang kit d&#39;extraction). Préchauffer le tampon d&#39;élution (AE) à 37 ° C afin d&#39;améliorer le rendement de l&#39;ADN élué. ADN masculins et féminins pour la courbe standard sont isolés de façon similaire. </li><li> (Facultatif) ADN génomique peut être encore purifié ou concentré selon la méthode de précipitation EtOH en présence d&#39;acétate de sodium 3 M (pH = 5,5). Les culots d&#39;ADN sont ensuite remises en suspension dans 40-50 ul d&#39;eau distillée pour l&#39;analyse immédiatement ou conservés à -20 ° C pour une utilisation ultérieure. </li><li> Mesurer la concentr ADNtion avec photomètre ND-1000 spectres Nanodrop. Les échantillons présentant des DO 260/280 entre 1,8 à 2,0 sont utilisés pour une analyse plus approfondie. </li><li> Diluer avec l&#39;ADN DNase sans H 2 O à 4ng/μl dans un volume total de 50 ul. </li></ol> 5. Préparation courbe standard Diluer l&#39;ADN masculin et féminin ADN à une concentration de 4 ng / ul, et rendre le mélange d&#39;ADN selon la Table1 <table border="1"><tbody><tr><td> % D&#39;ADN mâle en arrière-plan Femme </td><td> Volume (pi) de l&#39;ADN masculin 4ng/μl </td><td> Volume (pi) de l&#39;ADN femelle 4ng/μl </td><td> Le volume total (pl) </td></tr><tr><td> 0,2 </td><td> 1 </td><td> 499 </td><td> 500 </td></tr><tr><td> 0,5 </td><td> 1 </td><td> 199 </td><td> 200 </td></tr><tr><td> 2,5 </td><td> 5 </td><td> 195 </td><td> 200 </td></tr><tr><td> 12,5 </td><td> 25 </td><td> 175 </td><td> 200 </td></tr><tr><td> 50 </td><td> 100 </td><td> 100 </td><td> 200 </td></tr><tr><td> 87,5 </td><td> 175 </td><td> 25 </td><td> 200 </td></tr><tr><td> 100 </td><td> 200 </td><td> 0 </td><td> 200 </td></tr></tbody></table> La préparation des échantillons tableau 1. Pour la courbe standard. Normales hommes et femmes FVB / NJ souris à l&#39;âge de 8-12 sem ont été sacrifiés. Les cellules sanguines et BM ont été recueillies. ADN provenant de cellules mâles et des cellules femelles ont été isolées et remises en suspension à une concentration de 4ng/μl. Les ADN masculins et féminins ont été mélangés à des taux différents pour générer les mélanges d&#39;ADN d&#39;échantillons standard. 6. PCR en temps réel <ol><li> Mettre en place la plaque de réaction PCR en mélangeant SYBR Green Supermix réactif avec des amorces et l&#39;ADN génomique. Le volume de réaction (20 pi) contient 400 nM de chaque amorce et 5 pi d&#39;ADN génomique de cellules sanguines (4ng/μl x5 pi = 20 ng) dans chaque réaction. ADN samples normes et ont été établis en trois exemplaires. Les séquences d&#39;amorces sont présentés dans le tableau 2. </li></ol><table border="1"><tbody><tr><td> Nom de gène </td><td> Avant </td><td> Inverser </td></tr><tr><td> Bcl2 </td><td> 5&#39;-AAGCTGTCACAGAGGGGCTA </td><td> 5&#39;-CAGGCTGGAAGGAGAAGATG </td></tr><tr><td> Zfy1 </td><td> 5-TGGAGAGCCACAAGCTAACCA </td><td> 5&#39;-CCCAGCATGAGAAAGATTCTTC </td></tr></tbody></table> Tableau 2. Séquence de l&#39;amorce de RT-PCR pour Bcl2 murin et Zfy1. <ol start="2"><li> Effectuer la réaction de PCR en utilisant Biorad iQ5 machine de PCR dans les conditions suivantes: 95 ° C 3 min, 42 cycles d&#39;amplification de 95 ° C pendant 10 sec, 58 ° C pendant 25 s et 72 ° C pendant 15 s, suivi d&#39;une courbe de fusion- l&#39;étape. </li><li> Obtenir cycle seuil (Ct) des valeurs par Bio-Rad iQ5 2.1 Système Standard Edition optique. Calculer δCt (Ct Zfy1 &lt;/ Sup&gt; Ct-Bcl2) valeur, et Zfy1 niveau d&#39;expression est calculée en tant que valeur de 2-δ Ct. </li><li> Établir des courbes d&#39;étalonnage pour chaque série de réactions utilisant du 2-δCt lecture à partir connues mâle / femelle mélanges étalons. Les courbes d&#39;étalonnage sont générées par le moyen de pointage 2-δCt valeur de triplicatas à l&#39;ADN mâle% connue dans le mélange avec ajustement de la régression linéaire. </li></ol>

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Nous n&#39;avons pas d&#39;intérêts financiers concurrents de divulguer.

L&#39;objectif de notre étude est de fournir aux spectateurs une technique basée sur la PCR pour quantifier la prise de greffe de cellule donneuse dans un modèle concurrentiel osseuse murine greffe de moelle. Plusieurs études ont été rapportés par RT-PCR pour détecter les cellules du donneur dans des modèles de transplantation 9-10. Le groupe du Dr Schwarzenberger a d&#39;abord développé un modèle murin de greffe de moelle osseuse en utilisant PCR en temps réel pour amplifier chromosome Y spécif...

Murin moelle osseuse (MO) transplantation modèle a été développé dans les années 1960 1. Ce modèle a été largement utilisé pour l&#39;étude des cellules souches donateurs hématopoïétiques (CSH) la biologie dans une souris hôte receveur. Murin modèle de greffe de moelle osseuse nous a fourni de précieuses connaissances sur les fonctions HSC et de leur régulation et est indispensable dans la recherche HSC. Allogénique modèle greffe de moelle osseuse comme C57BL/6J H2b-souris Balb / C H2d ou d&#39;un modèle de transplantation congéniques CD45.2 comme C56Bl/6J-B6.SJL CD45.1 sont utilisés dans de nombreux laboratoires pour étudier la fonction des gènes sur l&#39;activité HSC 2, effet de traitement de la toxicomanie sur les maladies de fonction 3 ou greffe de CSH connexes tels que la réaction du greffon contre l&#39;hôte (GvHD) 4. Marqueurs de surface cellulaire tels que haplotype CMH ou CD45.1 sont couramment utilisés pour distinguer les donneurs de cellules dérivées de cellules de destinataire origine. C57BL/6J H2b, CD45.2 </sup&gt;, Balb / C H2d et B6.SJL CD45.1 sont les souches de souris les plus couramment utilisés dans la greffe de moelle osseuse, car la contribution cellule donneuse peut être facilement évaluée par cytométrie en flux mesure CD45.1 vs vs CD45.2 ou H2b H2D. Cependant, de nombreuses autres souches telles que FVB / NJ 5 et C3H sont également souvent utilisé pour générer transgénique ou génétiquement des souris knockout. Ces souris peuvent être recroisé à une lignée consanguine et maintenus dans un mélange génétique / CMH arrière-plan. Dans ces cas, la détermination de la prise de greffe de cellule donneuse et la fonction HSC pourrait être difficile en tant que marqueurs de surface spécifiques des donneurs de cellules peuvent ne pas être disponibles. Utilisation du chromosome Y spécifique sonde d&#39;ADN pour détecter les cellules du donneur mâle par transfert de Southern de sexe dépareillés greffe de moelle osseuse a été développé par le groupe du Dr Miwa 6. Ensuite, une PCR en temps réel pour la région Y détermination du sexe a été jugée une méthode précise et hautement spécifique pour quantifier macellules fœtales LE dans le système sanguin maternel 7. Ce concept a été adapté par le groupe du Dr Schwarzenberger pour le développement d&#39;une technique de PCR en temps réel dans un modèle de transplantation de moelle osseuse murine pour déterminer la prise de greffe de cellule donneuse 8. Nous avons également modifié cette méthode pour la mesure de la prise de greffe de cellule donneuse dans FVB / NJ modèle osseuse de souris greffe de moelle. Cette méthode est actuellement largement utilisés dans notre groupe pour étudier le rôle de la kinase PIM1 en biologie HSC.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> RPMI 1640 </td><td> Hyclone </td><td> SH30255.01 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-017 </td><td> Inactivé par la chaleur </td></tr><tr><td> Le chlorure d&#39;ammonium </td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td> Bicarbonate de potassium </td><td> Pêcheur </td><td> P184-500 </td><td></td></tr><tr><td> QIAamp d&#39;ADN de sang minikit </td><td> Qiagen </td><td> 51106 </td><td></td></tr><tr><td> Tamis cellulaire </td><td> BD biosciences </td><td></td><td></td></tr><tr><td> iQ SYBR Green Supermix </td><td> Biorad </td><td> 170-8882 </td><td></td></tr><tr><td> iQ5 temps réel machine PCR </td><td> Biorad </td><td></td><td></td></tr><tr><td> Spectra photomètre </td><td> Nanodrop ND-1000 </td><td> ND-1000 </td><td></td></tr></tbody></table>

using quantitative real-time pcr to determine donor cell engraftment in a competitive murine bone marrow transplantation model

Modèles murins de greffe de moelle osseuse constituent un outil important pour mesurer de cellules souches hématopoïétiques (CSH) et les gènes qui déterminent les fonctions / molécules qui régulent la CSH. Dans ces systèmes, le modèle de transplantation, la fonction des cellules souches hématopoïétiques est déterminé par la capacité de ces cellules à des souris greffer et reconstituer bénéficiaires irradiées de façon létale. Communément, la cellule donneuse contribution / prise de greffe est mesurée par des anticorps spécifiques aux bailleurs de protéines de surface cellulaire par cytométrie en flux. Cependant, cette méthode dépend fortement de la spécificité et de la capacité du marqueur de surface cellulaire pour différencier des bailleurs de fonds des cellules dérivées de cellules destinataire-nées, qui peuvent ne pas être disponibles pour toutes les souches de souris. Compte tenu de la diversité des souches de souris génétiquement modifiées sur le marché, cette surface cellulaire / cytométrie de flux basé sur la méthode comporte des limites importantes en particulier dans les souches de souris qui n&#39;ont pas bien définis marqueurs de surface de cellules du donneur séparer congéniques destinataire CElls. Ici, nous avons enregistré une technique basée sur la PCR pour déterminer la prise de greffe de cellule donneuse / contribution à des souris receveuses de transplantation. Nous avons transplanté hommes CSH donneurs de moelle osseuse de souris irradiées de façon létale congéniques femmes. Échantillons de sang périphérique ont été prélevés au moment de post-transplantation différents points. Des échantillons de moelle osseuse ont été obtenus à la fin des expériences. L&#39;ADN génomique a été isolé et le gène du chromosome Y spécifique, Zfy1, a été amplifié par PCR en temps réel quantitative. La prise de greffe des hommes donneurs de cellules dérivées des souris receveuses femelles a été calculé par rapport à la courbe standard avec pourcentage connu des ADN masculins vs femme. Bcl2 a été utilisé comme gène de référence pour normaliser la quantité d&#39;ADN total. Nos données suggèrent que cette approche permet de déterminer de manière fiable la prise de greffe de cellule donneuse et fournit une méthode utile, mais simple dans la mesure de la reconstitution hématopoïétique dans des modèles murins de transplantation de moelle osseuse. Notre méthode peut être effectué systématiquement dans la plupart des laboratoires, car aucunéquipements coûteux comme la cytométrie de flux est nécessaire.

Figures 1 et 2 montrent des exemples de courbes de référence tracées avec des valeurs moyennes de 2-δCt contre pourcentages d&#39;ADN masculin. Figure 1A montre la température de fusion spécifique pour Bcl2 et Zfy1 amplicons localisés à 78,5 ° C et 88,5 ° C respectivement. Bcl2 est utilisé en tant que gène de référence pour normaliser la quantité totale d&#39;ADN chargé dans chaque réaction de PCR. Bcl2 courbes d&#39;amplification (vue Log) pour chaque échan...

Watch this Scientific Journal Video about Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model at JoVE.com

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Détermination de la prise de greffe de cellule donneuse présente un défi dans des modèles de souris moelle osseuse greffe qui n&#39;ont pas bien définis marqueurs phénotypiques. Nous avons décrit une méthodologie pour quantifier les hommes greffe de cellule donneuse dans des souris femelles bénéficiaires de greffes. Cette méthode peut être utilisée dans toutes les souches de souris pour l&#39;étude des fonctions HSC.

Utilisation quantitative PCR en temps réel pour déterminer prise de greffe de donneurs de cellules dans un modèle murin concurrentiel greffe de moelle osseuse

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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15.3K vues.  Medical University of South Carolina. Détermination de la prise de greffe de cellule donneuse présente un défi dans des modèles de souris moelle osseuse greffe qui n'ont pas bien définis marqueurs phénotypiques. Nous avons décrit une méthodologie pour quantifier les hommes greffe de cellule donneuse dans des souris femelles bénéficiaires de greffes. Cette méthode peut être utilisée dans toutes les souches de souris pour l'étude des fonctions HSC.

Vidéo: Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

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 modèle murin de 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 ...

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Médecine

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Détection MicroRNA dans les tumeurs prostatiques par quantitative PCR en temps réel (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Évaluation pré-clinique des inhibiteurs de tyrosine kinase pour le traitement de la leucémie aiguë

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murin col de l&#39;utérus Cœur, la transplantation en utilisant une technique de modification de manchette

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Tissue Culture Modèle tridimensionnel pour étudier primaire moelle osseuse humaine et de ses affections malignes

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Liquide céphalo-rachidien profilage de microARN Utilisation quantitative PCR en temps réel

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murin de transplantation de la cornée: Un modèle pour étudier la forme la plus courante de la transplantation d&#39;organes solides

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Évaluation des métastases pulmonaires chez la souris tumeur mammaire modèles par PCR quantitative en temps réel

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modèle de transplantation aortique cervicale de Murine utilisant une technique modifiée de manchette de non-suture

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Analyse de cytométrie de flux des sous-ensembles de cellules immunitaires dans la rate murine, la moelle osseuse, les ganglions lymphatiques et le tissu synovial dans un modèle d’arthrose