Agradecemos ao Dr. Charles Greenberg para o uso de sua máquina do tempo real PCR. Este trabalho é apoiado por MUSC Hollings Cancer Center Startup Fundo, Hollings Cancer Center ACS IRG, ASCO Conquer Cancer Foundation Award Desenvolvimento de Carreira, 1K08HL NIH 103.780-01A1, e NIH 3P30CA138313-01S3. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente a posição oficial do Instituto Nacional de Saúde ou agentes de outros financiamentos.

1. Isolamento das células da medula óssea <ol><li> Euthanize masculinas dadoras FVB / NJ ratos fêmeas e FVB / NJ camundongos utilizando CO método 2, seguido de deslocação cervical. As fêmeas FVB / NJ células da medula óssea será utilizado como células competitivos. </li><li> Use pequenas tesouras e pinças, dissecar fêmures e tibiaes de ratos e coloque-os em um prato de 60 milímetros de cultura de tecido contendo 6 ml RPMI1640 gelada com 5% de calor FBS inativado. Use tecido Kimwipe para remover o músculo e outros tecidos. Cortar ambas as extremidades de cada eixo do osso no prato. </li><li> Ligar a extremidade do osso, com agulha 23G, em 3 de seringa cc, expulsar medula óssea com RPMI 1640 com 5% de FBS inactivado pelo calor para dentro do prato. Desagregar os tecidos da medula óssea por aspiração repetida usando a mesma agulha. Transferir a suspensão de células para 15 ml de tubo de centrífuga. </li><li> Girar as células durante 5 minutos a 400 xg, remover o sobrenadante, ressuspender as células em 1 ml de tampão de sangue vermelho quarto temperatura lise celular (155 mM pbicarbonato otassium, 10 mM de cloreto de amónio, 0,1 mM de EDTA, pH = 7,4) e incuba-se à temperatura ambiente durante 5 min, em seguida, adicionar 5-10 ml de meio RPMI 1640 com 5% de FBS inactivado pelo calor. </li><li> Passar as células através de um coador de células. Recolher o fluxo através de um tubo novo. Girar durante 5 minutos a 400 x g. Remover o sobrenadante, o sedimento de células não devem conter qualquer cor vermelha. A ausência de cor vermelha indica a remoção completa das células vermelhas do sangue. Ressuspender o sedimento de células em 10 ml de RPMI 1640 com 5% de FBS inactivado pelo calor. Vortex suavemente para assegurar que a suspensão celular é completamente uniforme. Tome uma alíquota e contar as células em um hemocitômetro. Calcular a quantidade de volume de células necessárias para o transplante de medula óssea e células de alíquotas suficientes e misturar células dadoras masculinas com células concorrente do sexo feminino, numa relação de 5:2. Girar durante 5 minutos a 400 xg, lavar com PBS, ressuspender em PBS com uma concentração final de células de dador a 5 x 10 células de 6 ml / e concorrente de 2 × 10 6 </sup&gt; / ml. </li></ol> 2. Transplante de Medula Óssea competitivo <ol><li> Irradiar ratos fêmeas receptoras (8-12 semanas de idade) com 137Cs radiador raios gama a uma dose única de 11Gy 4-6 hr antes do transplante de medula óssea. </li><li> Coloque os ratos irradiados do sexo feminino em um limitador mouse. Injectar o doador e mista células concorrentes via veia da cauda, ​​em 0,1 ml de volume total de tal forma que cada rato recebe 5 x 10 5 células dadoras e 2 × 10 5 concorrente células da medula óssea. </li></ol> 3. Coleta de Amostras Semana de casais após transplante de medula óssea, recolher amostras de sangue periférico (~ 50 ul) de rato receptor fêmea por sangramento retro-orbital sob anestesia condição. As amostras são recolhidas em tubos com EDTA revestidos. BM amostras também podem ser recolhidas no fim da experiência, geralmente, pelo menos, 4 meses pós-transplante, com procedimentos idênticos aos descritos anterior (Etapa 1), geralmente 20-40% de1 células BM tíbia são suficientes para tornar a quantidade suficiente de DNA. Amostras de sangue ou BM de idade correspondentes normais ratos fêmeas e machos também são coletadas para preparar curva padrão. 4. Isolamento de DNA genómico <ol><li> Adicionar volume 4 × de temperatura ambiente tampão de lise (~ ul 200) para cada amostra de sangue. Misturar bem e incubar à temperatura ambiente durante 5 min. Adicionar 1 ml de PBS e, em seguida girar para baixo para remover a maior parte do GV ​​lisada. </li><li> Isolar o ADN utilizando um estojo de extracção de ADN de sangue (DNA QIAmp kit de extracção de sangue). Pré-aquecer o tampão de eluição (AE), a 37 ° C para aumentar o rendimento do ADN eluído. DNAs masculinos e femininos para a curva padrão são isoladas da mesma forma. </li><li> (Opcional) O DNA genómico pode ser ainda purificado ou concentrados utilizando EtOH método de precipitação na presença de acetato de sódio 3 M (pH = 5,5). Os sedimentos de DNA são então ressuspensas em 40-50 ul de água destilada, para análise imediatamente ou armazenados a -20 ° C para uso futuro. </li><li> Medir o DNA Concentrção com Nanodrop fotômetro espectros ND-1000. As amostras com DO 260/280 entre 1,8-2,0 são utilizadas para análise posterior. </li><li> Diluir com DNAase DNA livre H 2 O para 4ng/μl em um volume total de 50 ul. </li></ol> 5. Preparação da curva padrão Diluir DNA do sexo masculino e feminino de ADN a uma concentração de 4 ng / ul, e faz-se a mistura de ADN de acordo com a Tabela 1 <table border="1"><tbody><tr><td> % De ADN masculino no fundo feminino </td><td> Volume (uL) de DNA macho 4ng/μl </td><td> Volume (uL) de DNA fêmea 4ng/μl </td><td> Volume total (pi) </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> Tabela A preparação da amostra 1. De curva padrão. Normais machos e fêmeas FVB / NJ ratos na idade de 8-12wks foram sacrificados. As células do sangue e da BM foram coletados. Os DNAs das células do sexo masculino e do sexo feminino células foram isoladas e re-suspensas numa concentração de 4ng/μl. Os DNAs de ambos os sexos foram misturadas em várias proporções para produzir as misturas de ADN de amostra padrão. 6. PCR em tempo real <ol><li> Configure a placa de reação de PCR pela mistura reagente SYBR Green Supermix com primers e DNA genômico. O volume de reacção (20 ul) contém 400 nM de cada iniciador e 5 ul de ADN genómico de células de sangue (uL 4ng/μl x5 = 20 ng), em cada reacção. DNA samples e padrões foram criados em triplicado. As sequências dos iniciadores são apresentadas na Tabela 2. </li></ol><table border="1"><tbody><tr><td> Nome do gene </td><td> Para a frente </td><td> Reverter </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> Tabela 2. Sequência do iniciador de RT-PCR para Bcl2 murino e Zfy1. <ol start="2"><li> Realizar PCR de reacção utilizando Biorad IQ5 máquina PCR com as seguintes condições: 95 ° C 3 min, 42 ciclos de amplificação de 95 ° C durante 10 seg, 58 ° C durante 25 seg e 72 ° C durante 15 segundos, seguido por uma fusão de curva passo. </li><li> Obter ciclo limiar (Ct) valores pelo Sistema IQ5 Bio-Rad Edition 2.1 ótico padrão. Calcular δCt (Ct Zfy1 &lt;/ Sup&gt;-Ct Bcl2), valor e Zfy1 nível de expressão é calculado como o valor de 2-δ Ct. </li><li> Estabelecer curvas padrão para cada uma das séries de reacção com 2-δCt leitura a partir da conhecida macho / fêmea misturas padrão. As curvas padrão são geradas através da representação gráfica da média de 2-δCt valor de triplicados para a DNA conhecido macho% na mistura com encaixe de regressão linear. </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>

Nós não temos nenhuma competição de interesses financeiros de divulgar.

O objetivo de nosso estudo é o de fornecer as audiências uma técnica de PCR baseado quantificar o enxerto de células de doadores em um modelo murino de osso competitivo transplante de medula. Vários estudos têm sido relatados por meio de RT-PCR para detectar as células de dadores em modelos de transplante 9-10. O grupo do Dr. Schwarzenberger a primeira desenvolveu um modelo murino óssea transplante de medula usando PCR em tempo real para amplificar cromossomo Y específico 8. Este método f...

Murino de medula óssea (BM) modelo de transplante foi desenvolvido pela primeira vez em 1960 1. Este modelo tem sido utilizado extensivamente para o estudo do dador de células estaminais hematopoiéticas (HSC) Biologia em um rato receptor hospedeiro. Murino osso modelo de transplante de medula nos proporcionou conhecimentos valiosos sobre as funções do HSC e sua regulação e é indispensável na pesquisa HSC. Alogénica modelo de transplante de medula óssea como C57Bl/6J H2b-Balb / C H2d ou modelo de transplante Congênicos como C56Bl/6J CD45.2-B6.SJL CD45.1 são usados ​​em muitos laboratórios, para estudar a função de genes em actividade HSC 2, efeito de tratamento com medicamentos para as doenças de função HSC 3 ou afins, tais como o transplante de enxerto-versus-hospedeiro (GVHD) 4. Marcadores de superfície celular tais como o haplotipo MHC ou CD45.1 são comumente usados ​​para distinguir células derivadas de dador de células-receptor originaram. C57Bl/6J H2b, CD45.2 </sup&gt;, Balb / C e H2d B6.SJL CD45.1 são as estirpes de ratos mais utilizados no transplante de medula óssea porque a contribuição das células do doador podem ser facilmente avaliadas por citometria de fluxo, medindo CD45.1 vs CD45.2 ou H2b vs H2D. No entanto, muitas outras cepas, como FVB / NJ 5 e C3H também são frequentemente utilizados para gerar transgênico ou geneticamente camundongos knockout. Estes ratos podem ser retrocruzada com uma linha pura e mantidos num fundo genético / MHC misto. Nestes casos, determinar o enxerto dador de células e função HSC pode ser difícil como marcadores específicos de dadores de células superficiais podem não estar disponíveis. Usando Y-cromossoma sonda específica de DNA para detectar as células de doadores do sexo masculino por Southern blot em sexo incompatíveis transplante de medula óssea foi desenvolvido pelo grupo do Dr. Miwa 6. Em seguida, um PCR em tempo real para a região Y-sexo determinação verificou-se ser um método preciso e altamente específico para quantificar macélulas fetais le no sistema de sangue materno 7. Este conceito foi adaptado pelo grupo do Dr. Schwarzenberger para o desenvolvimento de uma técnica de PCR em tempo real em um modelo murino de transplante de medula óssea para determinar o enxerto doador de 8 células. Nós ainda modificada deste método para a medição do enxerto dador de células no modelo do rato FVB / NJ transplante de medula óssea. Este método é actualmente utilizada extensivamente no nosso grupo para o estudo do papel da quinase Pim1 em biologia HSC.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </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> Inactivado por calor </td></tr><tr><td> Cloreto de amônio </td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td> Bicarbonato de potássio </td><td> Pescador </td><td> P184-500 </td><td></td></tr><tr><td> QIAamp Sangue DNA minikit </td><td> Qiagen </td><td> 51106 </td><td></td></tr><tr><td> Coador celular </td><td> BD biociência </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 tempo real máquina de PCR </td><td> Biorad </td><td></td><td></td></tr><tr><td> Spectra fotômetro </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

Murinos osso modelos de transplante de medula fornecer uma ferramenta importante na medição de células-tronco hematopoiéticas (HSC) funções e os genes que determinam / moléculas que regulam HSCs. Nestes sistemas modelo de transplante, a função de HSCs é determinada pela capacidade destas células para enxertar e reconstituir ratinhos receptores letalmente irradiados. Comummente, a célula doadora contribuição / enxerto é medida por meio de anticorpos específicos para o doador de proteínas da superfície celular, utilizando citometria de fluxo. No entanto, este método depende muito da especificidade e a capacidade do marcador de superfície celular de diferenciar células derivadas de dador a partir de células-receptor originadas, que podem não estar disponíveis para todas as estirpes de ratinho. Considerando os vários fundos de linhagens de camundongos geneticamente modificados no mercado, esta superfície celular / citometria de fluxo baseado em método tem limitações significativas, especialmente em linhagens de camundongos que não têm bem definidos marcadores de superfície de células de doadores separar Congênicos destinatário cells. Aqui, nós relatamos uma técnica baseada em PCR para determinar o enxerto de células do doador / contribuição em camundongos receptores de transplantes. Nós transplantadas machos dadores de medula óssea para HSCs letalmente irradiados congénicos ratinhos fêmea. Amostras de sangue periférico foram colhidas em diferentes pontos do tempo pós-transplante. As amostras de medula óssea foram obtidas no final das experiências. O ADN genómico foi isolado e o gene cromossoma Y específica, Zfy1, foi amplificado utilizando PCR quantitativa em tempo real. O enxerto do doador masculinos derivados de células nos ratinhos receptores do sexo feminino foi calculado contra curva padrão com a percentagem conhecida de DNAs macho versus fêmea. Bcl2 foi usado como um gene de referência para normalizar a quantidade total de ADN. Os nossos dados sugerem que esta abordagem fiável determina o enxerto de células do doador e proporciona um método útil, contudo simples para medir a reconstituição de células hematopoiéticas, em modelos de transplante de osso murino medula. O nosso método pode ser realizado rotineiramente em muitos laboratórios, porque nenhumequipamento caro, tais como citometria de fluxo é necessária.

Figura 1 e 2 apresentaram exemplos de curvas padrão traçada com valores médios de 2-δCt contra percentagens de ADN masculino. Figura 1A mostraram temperatura de fusão específica para Bcl2 e Zfy1 amplicons localizadas a 78,5 ° C e 88,5 ° C, respectivamente. Bcl2 é utilizado como um gene de referência para normalizar a quantidade total de ADN carregado em cada reacção de PCR. Bcl2 curvas de amplificação (ver Log) para cada amostra, padrão fundem uns com os outros...

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

Determinando o enxerto de células do doador representa um desafio em ratinho ósseas modelos de transplante de medula que carecem bem definidos marcadores fenotípicos. Nós descrevemos uma metodologia para quantificar o enxerto de células de doadores do sexo masculino em camundongos fêmeas receptoras de transplante. Este método pode ser utilizado em todas as estirpes de ratinho para o estudo de funções HSC.

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

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

using-quantitative-real-time-pcr-to-determine-donor-cell-engraftment

Research

JoVE Journal

Medicine

15.3K visualizações.  Medical University of South Carolina. Determinando o enxerto de células do doador representa um desafio em ratinho ósseas modelos de transplante de medula que carecem bem definidos marcadores fenotípicos. Nós descrevemos uma metodologia para quantificar o enxerto de células de doadores do sexo masculino em camundongos fêmeas receptoras de transplante. Este método pode ser utilizado em todas as estirpes de ratinho para o estudo de funções HSC.

Vídeo: 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

Um modelo do rato de No Utero Transplante

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

Medicina

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.

Detecção MicroRNA em tumores de próstata por Quantitative PCR em tempo real (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.

A avaliação pré-clínica da tirosina quinase inibidores para o tratamento de leucemia aguda

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.

Murino Cervical Modelo de coração Transplante usando uma técnica Cuff Modificado

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 Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

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.

Líquido cefalorraquidiano MicroRNA perfil usando Quantitative PCR em Tempo Real

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.

Murino transplante de córnea: Um modelo para estudar a forma mais comum de Transplante de Órgãos Sólidos

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.

Avaliação de metástases pulmonares em Rato mamárias tumorais Models por Quantitative PCR em tempo real

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 Modelo de Transplante de Aorta Cervical usando uma técnica de manguito não sutura modificada

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.

Análise de Citometria de Fluxo de Subconjuntos de Células Imunes dentro do Baço de Murina, Medula Óssea, Linfonodos e Tecido Sinovial em um Modelo de Osteoartrite