Wir danken Dr. Charles Greenberg für die Nutzung seiner Real-time PCR-Maschine. Diese Arbeit wird durch MUSC Hollings Cancer Center Gründerfonds, Hollings Cancer Center ACS IRG, ASCO Conquer Cancer Foundation Career Development Award, NIH 1K08HL 103.780-01A1 und NIH 3P30CA138313-01S3 unterstützt. Der Inhalt ist allein in der Verantwortung der Autoren und nicht unbedingt die offizielle Meinung der National Institutes of Health oder andere Mittel Agenten.

Ein. Bone Marrow Zellisolation <ol><li> Einschläfern männlichen Spenders FVB / NJ Mäusen und weibliche FVB / NJ Mäusen mit CO 2-Methode durch Genickbruch folgte. Die weiblichen FVB / NJ Knochenmarkzellen als wettbewerbsfähigen Zellen verwendet werden. </li><li> Verwenden Sie kleine Schere und Pinzette, sezieren Sie Oberschenkel und tibiaes von Mäusen und legen Sie sie in einem 60 mm Gewebekulturschale mit 6 ml eiskaltem RPMI1640 mit 5% hitzeinaktiviertem FBS. Verwenden Kimwipe Gewebe Muskeln und anderen Geweben zu entfernen. Schneiden Sie die beiden Enden jedes Knochens Welle in die Schüssel. </li><li> Verbinden Sie das Ende des Knochens mit 23G Nadel auf 3 cc Spritze spülen Knochenmark mit RPMI1640 mit 5% hitzeinaktiviertem FBS in die Schüssel. Disaggregieren Knochenmark Gewebe durch wiederholte Bestrebungen mit der gleichen Nadel. Übertragen der Zellsuspension zu 15 ml-Zentrifugenröhrchen. </li><li> Abzentrifugieren der Zellen für 5 min bei 400 × g, Entfernen des Überstandes werden die Zellen in 1 ml Raumtemperatur Erythrozyten-Lyse-Puffer (155 mM potassium Bicarbonat, 10 mM Ammoniumchlorid, 0,1 mM EDTA, pH = 7,4) und Inkubation bei Raumtemperatur für 5 min fügen 5-10 ml RPMI 1640 mit 5% Hitze-inaktiviertem FBS. </li><li> Führen Sie die Zellen durch eine Zelle Sieb. Sammeln des Durchflusses durch ein neues Röhrchen. Spindown für 5 min bei 400 x g beträgt. Entfernen Sie den Überstand; das Zellpellet sollten keine rote Farbe. Das Fehlen von roten Farbe eine vollständige Entfernung von roten Blutkörperchen. Zellpellet in 10 ml RPMI1640 mit 5% hitzeinaktiviertem FBS. Vorsichtig vortexen, um sicherzustellen, dass die Zellsuspension ist völlig einheitlich. Einen aliquoten und zählen Sie die Zellen in einem Hämazytometer. Berechnen Sie, wie viel Volumen der Zellen für Knochenmarktransplantation und aliquoten genug Zellen benötigt und mischen männlichen Spenderzellen mit Konkurrenten weiblichen Zellen bei einem Verhältnis von 5:2. Spindown für 5 min bei 400 × g, mit PBS gewaschen, in PBS resuspendieren mit der Endkonzentration von Donorzellen mit 5 × 10 6 / ml und Kompetitor Zellen bei 2 × 10 6 </sup&gt; / ml. </li></ol> 2. Competitive Bone Marrow Transplantation <ol><li> Bestrahlen weiblichen Empfängermäuse (8-12 wks alt) mit 137Cs Gammastrahlung Strahlers bei einer Einzeldosis von 11Gy 4-6 Std. vor Knochenmarktransplantation. </li><li> Legen Sie die bestrahlten weiblichen Mäusen in einem Maus-Verzögerer. Injizieren des gemischten Donor und Kompetitor-Zellen über die Schwanzvene in 0,1 ml Gesamtvolumen, so dass jede Maus 5 x 10 5 Spenderzellen und 2 × 10 5 Kompetitor Knochenmarkszellen empfängt. </li></ol> 3. Probenentnahme Paare Wochen nach Knochenmarktransplantation, sammeln peripheren Blutproben (~ 50 ul) von weiblichen Empfängermaus durch retro-orbitale Blutung unter Narkose Zustand. Die Proben werden in EDTA beschichtete Röhrchen gesammelt. BM Proben kann auch am Ende des Experiments gesammelt werden, in der Regel von mindestens 4 Monaten nach der Transplantation, mit denselben Verfahren wie vorangegangenen beschrieben (Schritt 1), in der Regel 20-40%1 Tibia BM-Zellen sind genug, um genügend DNA-Menge machen. Blut oder BM Proben aus gleichaltrigen normalen weiblichen und männlichen Mäusen werden auch gesammelt, um Standardkurve vorbereiten. 4. Isolierung genomischer DNA <ol><li> Add 4 × Volumen von Raumtemperatur RBC Lysispuffer (~ 200 ul) in jede Blutprobe. Gut mischen und Inkubation bei Raumtemperatur für 5 min. 1 ml PBS, dann drehen bis die meisten der lysierten RBC entfernen. </li><li> Isolieren Sie DNA mit einem Blut-DNA Extraktions-Kits (QIAmp DNA Blood Extraction Kit). Vorwärmen der Elutionspuffer (AE) bei 37 ° C, um die Ausbeute des eluierten DNA verstärken. Männliche und weibliche DNAs für Standard-Kurve ähnlich isoliert. </li><li> (Optional) Genomische DNA kann weiter gereinigt werden oder konzentriert mit EtOH Präzipitationsverfahren in Gegenwart von 3 M Natriumacetat (pH = 5,5). Die DNA-Pellets werden dann in 40-50 ul destilliertem Wasser zur Analyse resuspendiert sofort oder bei -20 ° C für zukünftige Verwendung. </li><li> Messen Sie die DNA konzentration mit Nanodrop ND-1000-Spektren Photometer. Proben mit OD 260/280 zwischen 1,8-2,0 für die weitere Analyse verwendet. </li><li> Verdünnen DNA mit DNase freie H 2 O in einem Gesamtvolumen von 50 ul 4ng/μl. </li></ol> 5. Standardkurve Vorbereitung Verdünnte männlichen und weiblichen DNA DNA mit einer Konzentration von 4 ng / pl, und stellen die DNA-Mischung gemäß den Tabelle1 <table border="1"><tbody><tr><td> % Der männlichen DNA in Female Hintergrund </td><td> Volumen (ul) 4ng/μl männliche DNA </td><td> Volumen (ul) 4ng/μl weibliche DNA </td><td> Gesamtvolumen (ul) </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> Tabelle 1. Probenvorbereitung für die Standardkurve. Normale männliche und weibliche FVB / NJ Mäuse im Alter von 8-12wks geopfert wurden. Blood and BM-Zellen wurden gesammelt. DNAs aus männlichen und weiblichen Zellen Zellen wurden isoliert und erneut suspendiert in einer Konzentration von 4ng/μl. Die männlichen und weiblichen DNAs wurden in verschiedenen Verhältnissen gemischt, um die DNA-Standardprobe Mischungen erzeugen. 6. Echtzeit-PCR <ol><li> Richten Sie die PCR-Reaktion Platte durch Mischen SYBR Green supermix Reagenz mit Primer und genomische DNA. Das Reaktionsvolumen (20 ul) enthält 400 nM von jedem Primer und 5 ul Blutkörperchen genomische DNA (4ng/μl x5 ul = 20 ng) in jeder Reaktion. DNA samples und Standards wurden in dreifacher Ausführung gesetzt. Die Sequenzen der Primer sind in Tabelle 2 gezeigt. </li></ol><table border="1"><tbody><tr><td> Gene Namen </td><td> Vorwärts </td><td> Umkehren </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> Tabelle 2. RT-PCR-Primersequenz für murines und Bcl2 Zfy1. <ol start="2"><li> Führen PCR-Reaktion mit Biorad iQ5 PCR-Maschine mit den folgenden Bedingungen: 95 ° C, 3 Min., 42 Amplifikationszyklen bei 95 ° C für 10 sec, 58 ° C für 25 sec und 72 ° C für 15 s, durch ein Schmelz-Kurve folgt fort. </li><li> Erhalten Zyklus Schwelle (Ct)-Werte von Bio-Rad iQ5 2,1 Standard Edition Optical System. Calculate ACt (Ct Zfy1 &lt;/ Sup&gt;-Ct Bcl2) Wert und Zfy1 Expressionsniveau wird als der Wert von 2-δ Ct berechnet. </li><li> Stellen Standardkurven für jede Reaktion Serie mit 2-ACt Lesung aus bekannten männlichen / weiblichen Standard-Mischungen. Die Standardkurven werden durch Auftragen der mittleren von 2-ACt Wert Triplikaten dem bekannten% männlich-DNA im Gemisch mit linearen Regression Beschlag erzeugt. </li></ol>

<ol>
	<li>McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+radiation+sensitivity+of+normal+mouse+bone+marrow+cells,+determined+by+quantitative+marrow+transplantation+into+irradiated+mice.">The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice.</a> Radiat. Res. 13, 115-125 (1960).</li><li>Xiao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+lacking+Ott1+display+aspects+associated+with+aging+and+are+unable+to+maintain+quiescence+during+proliferative+stress.">Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress.</a> Blood. 119, 4898-4907 (2012).</li><li>Kang, Y., Chen, B. J., Deoliveira, D., Mito, J., Chao, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+enhancement+of+donor+hematopoietic+cell+engraftment+by+the+CXCR4+antagonist+AMD3100+in+a+mouse+transplantation+model.">Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.</a> PLoS One. 5, e11316 (2010).</li><li>Sadeghi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GVHD+after+chemotherapy+conditioning+in+allogeneic+transplanted+mice.">GVHD after chemotherapy conditioning in allogeneic transplanted mice.</a> Bone Marrow Transplant. 42, 807-818 (2008).</li><li>Taketo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FVB/N:+an+inbred+mouse+strain+preferable+for+transgenic+analyses.">FVB/N: an inbred mouse strain preferable for transgenic analyses.</a> Proc. Natl. Acad. Sci. U.S.A. 88, 2065-2069 (1991).</li><li>Morisaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+analysis+using+a+Y-chromosome-specific+probe+following+bone+marrow+transplantation.">Genotypic analysis using a Y-chromosome-specific probe following bone marrow transplantation.</a> Am. J. Hematol. 27, 30-33 (1988).</li><li>Lo, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+fetal+DNA+in+maternal+plasma+and+serum:+implications+for+noninvasive+prenatal+diagnosis.">Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.</a> Am. J. Hum. Genet. 62, 768-775 (1998).</li><li>Byrne, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimerism+analysis+in+sex-mismatched+murine+transplantation+using+quantitative+real-time+PCR.">Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.</a> Biotechniques. 32, 279-280 (2002).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+recipient-derived+cells+in+allograft+neointima+formation+and+the+response+to+stent+implantation.">Contribution of recipient-derived cells in allograft neointima formation and the response to stent implantation.</a> PLoS One. 3, e1894 (2008).</li><li>Bosio, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+real-time+quantitative+PCR+and+DNA+hybridization+for+quantitation+of+male+DNA+following+myoblast+transplantation.">A comparison between real-time quantitative PCR and DNA hybridization for quantitation of male DNA following myoblast transplantation.</a> Cell Transplant. 13, 817-821 (2004).</li><li>Merchant, A., Joseph, G., Wang, Q., Brennan, S., Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gli1+regulates+the+proliferation+and+differentiation+of+HSCs+and+myeloid+progenitors.">Gli1 regulates the proliferation and differentiation of HSCs and myeloid progenitors.</a> Blood. 115, 2391-2396 (2010).</li><li>Nagamine, C. M., Chan, K., Hake, L. E., Lau, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+candidate+testis-determining+Y+genes+(Zfy-1+and+Zfy-2)+are+differentially+expressed+in+fetal+and+adult+mouse+tissues.">The two candidate testis-determining Y genes (Zfy-1 and Zfy-2) are differentially expressed in fetal and adult mouse tissues.</a> Genes &amp; development. 4, 63-74 (1990).</li><li>Grundler, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+PIM+serine/threonine+kinases+in+FLT3-ITD-induced+leukemogenesis+reveals+PIM1+as+regulator+of+CXCL12-CXCR4-mediated+homing+and+migration.">Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration.</a> J. Exp. Med. 206, 1957-1970 (2009).</li></ol>

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Das Ziel unserer aktuellen Studie ist es, dem Publikum einen PCR-basierten Technik Donorzelle Transplantation in einem kompetitiven murine Knochenmark-Transplantation Modell zu quantifizieren. Mehrere Studien haben berichtet mittels RT-PCR, um Spenderzellen in der Transplantationsmedizin Modelle 9-10 zu erkennen. Dr. Schwarzenberger Die Gruppe entwickelte zunächst eine murine Knochenmark-Transplantation Modell mit real-time PCR zu amplifizieren y-Chromosom-spezifischen 8. Diese Methode wurde verwe...

Murine Knochenmark (BM) Transplantation Modell wurde erstmals in den 1960er Jahren 1 entwickelt. Dieses Modell wurde ausgiebig für das Studium der Donor hämopoetischen Stammzelle (HSC) Biologie in einem Wirt Empfängermaus verwendet. Murine Knochenmarktransplantation Modell hat uns mit wertvollen Wissen über HSC-Funktionen und deren Regulierung vorgesehen und in HSC Forschung unverzichtbar. Allogene Knochenmarktransplantation Modell wie C57Bl/6J H2b-Balb / C H2d oder kongene Transplantation Modell wie C56Bl/6J CD45.2-B6.SJL CD45.1 werden in vielen Laboren verwendet, um die Genfunktion auf HSC Aktivität 2 studieren, Wirkung Arzneimittelbehandlung auf HSC Funktion 3 oder Transplantation Krankheiten wie Graft-versus-host disease (GvHD) 4. Zelloberflächenmarker wie MHC Haplotyp oder CD45.1 werden häufig zur Unterscheidung von Donor-abgeleiteten Zellen vom Empfänger-Ursprung Zellen verwendet. C57Bl/6J H2b, CD45.2 </sup&gt; sind Balb / C H2d und B6.SJL CD45.1 die am häufigsten verwendeten Mausstämmen in Knochenmarkstransplantation, weil die Donorzelle Beitrag kann leicht durch Durchflußzytometrie gemessen CD45.1 vs CD45.2 oder H2b vs beurteilen H2D. Allerdings sind viele andere Stämme, wie FVB / NJ 5 und C3H auch häufig verwendet, um gentechnisch transgenen oder Knockout-Mäuse zu erzeugen. Diese Mäuse können zu einer Inzuchtlinie rückgekreuzt und gewartet werden in einer gemischten genetischen / MHC Hintergrund. In diesen Fällen könnte Bestimmen Donorzelle Engraftment und HSC Funktion schwierig sein als Donor-spezifische Zelloberflächenmarker nicht zugänglich. Mit Y-Chromosom-spezifischen DNA-Sonde, um die Donor männlichen Zellen durch Southern-Blot in sex-mismatched Knochenmarktransplantation erkennen wurde zuerst von Dr. Miwa der Gruppe 6 entwickelt. Dann wurde ein Echtzeit-PCR zur geschlechtsbestimmenden Bereich Y festgestellt eine genaue und hochspezifische Methode ma quantifizieren seinle fetalen Zellen im mütterlichen Blut System 7. Dieses Konzept wurde von Dr. Schwarzenbergers Gruppe für die Entwicklung eines Echtzeit-PCR-Technik in einem murinen Knochenmark-Transplantation Modell Donorzelle Engraftment 8 bestimmen angepasst. Wir weiter diese Methode für die Messung der Donorzelle Transplantation in FVB / NJ Maus Knochenmarktransplantation Modell modifiziert. Dieses Verfahren wird derzeit intensiv in unserer Gruppe für die Untersuchung der Rolle von Pim1 Kinase in HSC Biologie genutzt.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </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> Hitzeinaktiviertem </td></tr><tr><td> Ammoniumchlorid </td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td> Kaliumbicarbonat </td><td> Fischer </td><td> P184-500 </td><td></td></tr><tr><td> QIAamp DNA Blood minikit </td><td> Qiagen </td><td> 51106 </td><td></td></tr><tr><td> Zellsieb </td><td> BD Bioscience </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 real time PCR-Maschine </td><td> Biorad </td><td></td><td></td></tr><tr><td> Spectra Photometer </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

Murine Knochenmark-Transplantation Modelle bieten ein wichtiges Instrument bei der Messung hämatopoetischen Stammzellen (HSC)-Funktionen und die Bestimmung Gene / Moleküle, die HSCs regulieren. In diesen Transplantationsmodell Systemen wird die Funktion des HSK durch die Fähigkeit dieser Zellen, einprägen und rekonstituieren letal bestrahlten Mäusen Empfängers bestimmt. Üblicherweise wird die Donorzelle Beitrag / Verpflanzung durch Antikörper gegen Donor-spezifische Zelloberflächenproteine ​​mittels Durchflusszytometrie gemessen. Allerdings ist diese Methode hängt stark von der Spezifität und die Fähigkeit des Zelloberflächenmarkers zu Donor-abgeleiteten Zellen von Empfänger-Zellen stammt, die möglicherweise nicht für alle Maus-Stämmen zu unterscheiden. Betrachtet man die verschiedenen Hintergründen von gentechnisch veränderten Mauslinien auf dem Markt, hat diese Zelle Oberfläche / Durchflusszytometrie basierende Methode erheblichen Einschränkungen vor allem in Maus-Stämme, die gut definierte Oberfläche Marker fehlen, um getrennte Spenderzellen aus kongene Empfänger cells. Hier haben wir über einen PCR-basierten Technik Donorzelle Engraftment / Beitrag Transplantatempfänger Mäusen zu bestimmen. Wir transplantiert männlichen Spender-Knochenmark HSCs zu letal bestrahlten kongene weiblichen Mäusen. Periphere Blutproben wurden zu verschiedenen Zeitpunkten nach der Transplantation gesammelt. Knochenmark-Proben wurden am Ende der Experimente erhalten. Genomische DNA wurde isoliert und das Y-Chromosom spezifischen Gens, Zfy1, wurde amplifiziert mittels quantitativer Echtzeit-PCR. Die Verpflanzung von männlichen Spenders-abgeleiteter Zellen in den weiblichen Empfängermäuse wurde gegen Standardkurve mit bekannten Anteil männlicher vs weiblichen DNAs berechnet. Bcl2 wurde als Referenz-Gen um das Gesamt-DNA Menge normalisieren verwendet. Unsere Daten deuten darauf hin, dass dieser Ansatz zuverlässig bestimmt Donorzelle Verpflanzung und bietet eine nützliche, aber einfache Methode zur Messung hämatopoetischen Zellen Rekonstitution in murine Knochenmark-Transplantation Modelle. Unsere Methode kann routinemäßig in den meisten Labors durchgeführt werden, da keinekostspielige Ausrüstung wie Durchflusszytometrie erforderlich.

Abbildung 1 und 2 zeigten Beispiele von Standard-Kurven mit Mittelwerten von 2-ACt gegen Prozentangaben männlicher DNA. 1A zeigten spezifische Schmelztemperatur für Bcl2 und Zfy1 Amplikons bei 78,5 ° C und 88,5 ° C lokalisiert sind. Bcl2 wird als Referenz-Gen, um die Gesamtmenge der geladenen DNA in jeder PCR-Reaktion eingesetzt zu normalisieren. Bcl2 Amplifikationskurven (Log Ansicht) für jede Standardprobe miteinander unabhängig von männlichen DNA-Konzentration versc...

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

Bestimmung Donorzelle Transplantation stellt eine Herausforderung in der Maus Knochenmarktransplantation Modelle, die gut definierte phänotypische Marker fehlen. Wir beschrieben eine Methode zur männlichen Spenders Engraftment bei weiblichen Transplantatempfängers Mäusen zu quantifizieren. Dieses Verfahren kann in allen Mausstämmen zur Untersuchung von HSC-Funktionen verwendet werden.

Mittels quantitativer real-time PCR zum Donor Cell Engraftment in einem Competitive Murine Bone Marrow Transplantation Modell fest

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 Aufrufe.  Medical University of South Carolina. Bestimmung Donorzelle Transplantation stellt eine Herausforderung in der Maus Knochenmarktransplantation Modelle, die gut definierte phänotypische Marker fehlen. Wir beschrieben eine Methode zur männlichen Spenders Engraftment bei weiblichen Transplantatempfängers Mäusen zu quantifizieren. Dieses Verfahren kann in allen Mausstämmen zur Untersuchung von HSC-Funktionen verwendet werden.

Serie: 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

Einem Mausmodell der 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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Medizin

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.

MicroRNA-Detektion in Prostatatumoren durch 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 ...

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.

Präklinische Evaluierung von Tyrosinkinase-Inhibitoren für die Behandlung von akuter Leukämie

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.

Murine Cervical Herztransplantation Modell mithilfe eines modifizierten Cuff-Technik

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.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

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.

Liquor microRNA Profiling mittels quantitativer 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 ...

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.

Murine Hornhauttransplantation: Ein Modell, das die häufigste Form der Organtransplantation Studieren

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.

Beurteilung von Lungenmetastasen in Maus-Mammatumormodelle durch quantitative Echtzeit-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 ...

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.

Murine Cervical Aortic Transplantation Modell mit einer modifizierten Non-Suture Manschettentechnik

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.

Durchflusszytometrie Analyse von Immunzell-Subsets innerhalb der Murinenmilen, Knochenmark, Lymphknoten und Synovialgewebe in einem Osteoarthritis-Modell