Este estudo foi financiado pelo NIH R00HL102154, e um da American Society of Hematology prêmio estudioso a P Ji.

Os experimentos descritos neste protocolo foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Universidade Northwestern Animal Care Institucional e Comitê de Uso. 1 Preparação de Meio de Cultura <ol><li> Preparar a solução de fibronectina. Adicionar 1 ml de água para um frasco de fibronectina humana (1 mg). Deixar solução em cultura da capa de tecido, durante 30 minutos, sem agitação. Transferir o líquido total a 50 ml de PBS para fazer uma concentração final de 20 ug / ml e suavemente misturar bem. Adicione a solução de fibronectina fresco antes do revestimento da chapa (ver abaixo). </li><li> Prepare 50 ml de Epo meio livre (médio SCF). Combinar os ingredientes listados na Tabela 1. O meio pode ser armazenado a 4 ° C, durante 1 mês, quando feita. </li><li> Preparar 50 ml de Epo contendo meio. Combinar os ingredientes listados na Tabela 2. O meio pode ser armazenado a 4 ° C, durante 1 mês, quando feita. </li></ol&gt; 2. Fibronectina placa revestida <ol><li> Adicionar 1 ml de solução de fibronectina a cada poço de uma placa de 6 poços (ou 0,5 ml a cada poço de uma placa de 12 poços). Deixar a placa de cobertura na cultura de tecidos durante 1 h. </li><li> Lavar os poços duas vezes com água estéril e secar a placa. </li><li> Enrole a placa e guardar na câmara fria a 4 ° C. </li></ol> 3 Purificação de fígado fetal Erythroblasts NOTA: Use fígados fetais de E13 para E15 cronometrado camundongos grávidas (C57BL / 6). E13.5 fígados são preferidas para maximizar o rendimento de progenitores de CFU-E. Para obter as ratas grávidas cronometrados, 6-8 semanas de idade camundongos machos e fêmeas foram colocados em uma gaiola (geralmente um macho com uma ou duas mulheres). Tampões vaginais fêmeas foram verificadas na manhã seguinte para determinar se o acasalamento com sucesso. O primeiro dia de gestação foi no dia seguinte a ficha foi encontrada. <ol><li> Preparação das células totais de fígado fetal <ol><li> Sacrifique o mouse por inalação de CO 2 e deslocamento cervical. Pulverizar o abdômen com álcool 70% para desinfecção. Abra o abdômen com dissecando tesoura para remover o útero. Lavar o útero em PBS 1x. </li><li> Dissecar os fetos em condições estéreis, utilizando uma capa de cultura de tecidos e ferramentas autoclavados e lave em 1x PBS. </li><li> Dissecar os fígados fetais dos fetos. Segure o corpo do feto com um fórceps, retire com cuidado o fígado fetal (na cor rosa) de distância do corpo com outro fórceps. Limpe o fígado fetal com a pinça para remover os tecidos fibróticos associados. Coloque todos os fígados fetais frescos em 1x PBS contendo 10% de soro fetal bovino. </li><li> Mecanicamente perturbar fígados fetais pipetando para cima e para baixo. </li><li> Filtra-se a suspensão de células através de um coador de células 40 pm em um tubo de 50 ml. Agregar as células a 800 xg durante 5 min. </li><li> Aspirar o sobrenadante e ressuspender as células em 1 ml de PBS com FBS a 10% (cerca de 8 E13.5 fígados fetaispor ml). </li></ol></li><li> Purificação de Ter119 Erythroblasts negativos <ol><li> Bloquear as células com IgG de rato (0,2 mg / ml) em gelo durante 15 min. </li><li> Sem lavagem ou centrifugação, adicionar anticorpo anti-Ter119 biotinilado para a suspensão de células (1 ug / ml). Incubar durante 15 min em gelo. </li><li> Lavar com PBS 1x com 10% de FBS (1:10) e centrifugar a 800 xg durante 5 min. Ressuspender as células em 1 ml de PBS com 10% de FBS. </li><li> Adicionar 75 ul de estreptavidina conjugada com partículas magnéticas e incuba-se durante 10 minutos em gelo. </li><li> Adicionar o volume para 2,5 ml com PBS contendo 10% de FBS. Transferência da suspensão de células em 5 ml de um tubo de polipropileno de fundo redondo. </li><li> Inserir o tubo de um aparelho de triagem celular magnética e incubar durante 10 min. Células positivas Ter119 vai anexar ao lado do tubo. Os eritroblastos negativos Ter119 solta permanece na suspensão. </li><li> Despeje a suspensão de células em 5 ml de um novo tubo de polipropileno redondo inferior. </li><li><html> Repita os passos 3.2.6 e 3.2.7 para remover as células positivas para Ter119 residual. </li><li> Agregar as células a 800 xg durante 5 min. Aspirar o sobrenadante. Ressuspender as células em 1 ml de PBS contendo 10% de FBS e contagem de células viáveis ​​com azul de tripano. </li></ol></li></ol> 4. Transdução com vírus Encoding cDNA ou shRNA <ol><li> Placa as células fetais de fígado negativos Ter119 purificada a 3-5 x 10 5 / po de uma fibronectina revestido de 12 poços (ajustar o número de células se utilizando uma placa diferente). </li><li> Adicionar polibreno a uma concentração final de 10 ug / ml para facilitar a transdução virai. Rotação infectar as células com lentivírus ou retrovírus, o ADNc que codifica ou shRNA, a 800 xg durante 1-1,5 h a 37 ° C. </li></ol> 5. Cultura das transduzidas Ter119 Negative mouse fígado fetal Erythroblasts <ol><li> Para testar as funções dos genes na fase precoce da eritropoiese do terminal (Figura 1 as linhas a tracejado).&quot;&gt; </ol.><ol><li><ol. li=""> Cultura das células transduzidas em meio isento de Epo (SCF) médio durante 12 horas para permitir a expressão dos genes e manutenção do estado de progenitor transduzidas. </ol.></li><li> Centrifuga-se as células a 800 xg durante 5 min. Aspirar o sobrenadante. Ressuspender as células em 1 ml de Epo contendo o meio em cada poço de uma placa de 12 poços. </li><li> Depois da cultura durante 24 ou 48 horas, a colheita das células para outros ensaios. </li></ol></li><li> Teste as funções dos genes na fase final da eritropoiese de terminal (Figura 1 linhas sólidas). <ol><li> Cultura das células transduzidas imediatamente Epo contendo meio. </li><li> Depois da cultura durante 24 ou 48 horas, a colheita das células para os outros ensaios. </li></ol></li></ol> 6 A coloração das células cultivadas de fígado fetal para Análise Citometria de Fluxo <ol><li> Lave e ressuspender as células em 1x PBS. Colheita de 2 x 10 5 células em cultura para análise por citometria de fluxo. </li><li> Incubaras células com anticorpos fluoróforos, durante 20 minutos à temperatura ambiente no escuro. Para testar a diferenciação, utilizar APC anti-CD71 conjugado e conjugado com PE anti-Ter119. Para testar a enucleação, utilizar Hoechst 33342 mancha para além dos outros anticorpos. </li><li> Lavar as células com PBS 1x. Ressuspender as células em 0,5 ml de PBS contendo 1% de FBS e o iodeto de propídio (PI) (1 ug / ml) para corar as células mortas e as excluir análise. </li><li> Fluxo de Conduta análise cytrometric. Analise as células de controle de cores coradas e não coradas primeiro single para o ajuste e compensação do citômetro de fluxo. </li></ol>

<ol>
	<li>Hattangadi, S. M., Wong, P., Zhang, L., Flygare, J., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+stem+cell+to+red+cell:+regulation+of+erythropoiesis+at+multiple+levels+by+multiple+proteins,+RNAs,+and+chromatin+modifications.">From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications.</a> Blood. 118 (24), 6258-6268 (2011).</li><li>Richmond, T. D., Chohan, M., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+cells+red:+signal+transduction+mediated+by+erythropoietin.">Turning cells red: signal transduction mediated by erythropoietin.</a> Trends in cell biology. 15 (3), 146-155 (2005).</li><li>Eshghi, S., Vogelezang, M. G., Hynes, R. O., Griffith, L. G., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha4beta1+integrin+and+erythropoietin+mediate+temporally+distinct+steps+in+erythropoiesis:+integrins+in+red+cell+development.">Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development.</a> The Journal of cell biology. 177 (5), 871-880 (2007).</li><li>Koury, M. J., Bondurant, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+by+erythropoietin+of+viability+and+maturation+of+murine+erythroid+precursor+cells.">Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.</a> Journal of cellular physiology. 137 (1), 65-74 (1988).</li><li>Ji, P., Yeh, V., Ramirez, T., Murata-Hori, M., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+2+is+required+for+chromatin+condensation+and+subsequent+enucleation+of+cultured+mouse+fetal+erythroblasts.">Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts.</a> Haematologica. 95 (12), 2013-2021 (2010).</li><li>Dumitriu, 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=Sox6+cell-autonomously+stimulates+erythroid+cell+survival,+proliferation,+and+terminal+maturation+and+is+thereby+an+important+enhancer+of+definitive+erythropoiesis+during+mouse+development.">Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development.</a> Blood. 108 (4), 1198-1207 (2006).</li><li>Rector, K., Liu, Y., Van Zant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+hematopoietic+stem+cell+isolation+methods.">Comprehensive hematopoietic stem cell isolation methods.</a> Methods in molecular biology. 976, 1-15 (2013).</li><li>Rossi, L., Challen, G. A., Sirin, O., Lin, K. K., Goodell, M. A. <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+cell+characterization+and+isolation.">Hematopoietic stem cell characterization and isolation.</a> Methods in molecular biology. 750, 47-59 (2011).</li><li>Choi, H. S., Lee, E. M., Kim, H. O., Park, M. I., Baek, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+control+of+terminal+erythropoiesis+via+physical+interactions+among+erythroid+cells.">Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells.</a> Stem cell research. 10 (3), 442-453 (2013).</li><li>Bhatia, 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=Short-chain+fatty+acid-mediated+effects+on+erythropoiesis+in+primary+definitive+erythroid+cells.">Short-chain fatty acid-mediated effects on erythropoiesis in primary definitive erythroid cells.</a> Blood. 113 (25), 6440-6448 (2009).</li><li>Zhang, J., Socolovsky, M., Gross, A. W., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ras+signaling+in+erythroid+differentiation+of+mouse+fetal+liver+cells:+functional+analysis+by+a+flow+cytometry-based+novel+culture+system.">Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system.</a> Blood. 102 (12), 3938-3946 (2003).</li><li>Socolovsky, 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=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98 (12), 3261-3273 (2001).</li><li>Ji, P., Jayapal, S. R., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2.">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nature cell biology. 10 (3), 314-321 (2008).</li><li>Chida, D., Miura, O., Yoshimura, A., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytokine+signaling+molecules+in+erythroid+differentiation+of+mouse+fetal+liver+hematopoietic+cells:+functional+analysis+of+signaling+molecules+by+retrovirus-mediated+expression.">Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.</a> Blood. 93 (5), 1567-1578 (1999).</li><li>Patel, V. P., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fibronectin+matrix+is+required+for+differentiation+of+murine+erythroleukemia+cells+into+reticulocytes.">A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.</a> The Journal of cell biology. 105 (6 Pt 2), 3105-3118 (1987).</li><li>Udupa, K. B., Lipschitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-induced+suppression+of+erythropoiesis:+the+role+of+erythropoietin+and+a+heme+synthesis+stimulating+factor.">Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor.</a> Blood. 59 (6), 1267-1271 (1982).</li><li>Koury, M. J., Sawyer, S. T., Brandt, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+erythropoiesis.">New insights into erythropoiesis.</a> Current opinion in hematology. 9 (2), 93-100 (2002).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Aqui apresentamos um sistema único para analisar cronologicamente rato fetal eritropoiese terminal de fígado. Através da aplicação de diferentes condições de cultivo, dissecamos sucesso eritropoiese terminais em fases precoces e tardias. Isto é particularmente importante para determinar os mecanismos de genes com funções múltiplas. Por exemplo, GTPases Rac desempenham papéis importantes em diferentes estágios de eritropoiese terminal. Inibição de Rac GTPases na fase inicial de diferenciação e prolifera?...

Eritropoiese é o processo de diferenciação de células-tronco hematopoiéticas multipotentes para amadurecer eritrócitos. Este processo gradual inclui a formação de unidades formadoras empenhados explosão eritróide (UFB-ES), os que se dividem rapidamente unidades formadoras de colônias eritróides (UFC-ES), e morfologicamente eritroblastos reconhecíveis 1,2. Eritropoiese Terminal a partir de células progenitoras CFU-E envolve sequencial estágios independentes 2,3-dependente e eritropoietina. Na fase inicial da eritropoiese terminal, eritropoietina (Epo) se liga ao seu receptor na superfície da célula e induz uma série de vias de sinalização a jusante que previnem a apoptose celular e promovem a divisão celular rápida e de 1,4 a expressão do gene. No final da fase de eritropoiese terminal, eritroblastos submeter saída do terminal ciclo celular, condensação de cromatina e núcleo, e extrusão dos núcleos altamente condensadas 5. Nossa compreensão do terminal deeritropoiese tem melhorado muito nas últimas décadas, que é em grande parte devido ao sucesso do uso de vários in vitro e in vivo de mouse 6-9. Entre estes modelos, a cultura in vitro de rato fetal eritroblastos fígado proporciona muitas vantagens, incluindo a facilidade de purificação de células, proliferação e diferenciação rápida, e um mímico mais perto da eritropoiese humano 10,11. Neste sistema, um grande número de células progenitoras eritróides a partir de fígados de rato fetais pode ser facilmente isolado por purificação a única etapa de Ter119 (um marcador para as células eritróides maduras) eritroblastos negativos. Durante a cultura de dois dias dos eritroblastos, a diferenciação destas células podem ser monitorizados por uma análise de citometria de fluxo com base na expressão de superfície do receptor de transferrina (CD71) e o antigénio Ter119 12. Além disso, a enucleação dos eritroblastos de diferenciação terminal pode ser detectada por uma máquina de ADN (Hoechst 33342) 13. Além disso, as células progenitoras purificadas, pode ser geneticamente modificada por expressão de ADNc exógeno ou pequenas hairpin RNAs (shRNAs) para os genes de interesse, o que facilita os estudos mecanicistas das funções de expressão do gene na eritropoiese 11,13,14. Por outro lado, a rápida taxa de crescimento celular podem ser uma espada de dois gumes, uma vez que é difícil de caracterizar as funções de genes em diferentes fases da eritropoiese terminal. Na maioria dos casos, é difícil determinar se a funções de genes específicos na fase precoce da eritropoiese terminal de desde pelo tempo que o ADNc ou shRNAs expressa, as células já passou da fase precoce. Para resolver este problema, foi desenvolvido um sistema único para dissecar os estágios iniciais e tardios da eritropoiese terminal. Para a fase inicial da eritropoiese terminal, geneticamente modificados Ter119 eritroblastos negativas foram cultivadas em meio livre de Epo, mas que contém o factor de células estaminais (SCF), IL-6 e FLT3ligante para manter seu status progenitor e permitir que os cDNAs transduzidas ou shRNA a expressão 13. As células foram mudadas para meio contendo EPO depois de 12 horas para induzir a proliferação e diferenciação celular. Desta forma, quando as células se diferenciaram, os cDNAs foram transduzidas ou shRNAs já expressa. Para o estágio tardio da eritropoiese terminal, Ter119 eritroblastos negativas foram cultivadas em meio contendo EPO, imediatamente após a transdução. Portanto, pode-se analisar as funções dos genes de interesse na fase tardia da eritropoiese terminal. Em resumo, uma ampla aplicação deste sistema ajudaria funções dos genes dissecar em diferentes estágios de eritropoiese terminal.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

Eritropoiese envolve um processo dinâmico que começa com unidades formadoras empenhados explosão eritróide (UFB-ES) seguido de divisão rápida erythroid unidades formadoras de colônia (UFC-Es). Depois de CFU-Es, as células são morfologicamente reconhecíveis e geralmente denominado eritroblastos terminais. Um dos desafios para o estudo da eritropoiese terminal é a falta de abordagens experimentais para dissecar as funções dos genes de uma forma cronológica. Neste protocolo, descrevemos uma estratégia única para determinar as funções dos genes nas fases precoces e tardias da eritropoiese terminal. Neste sistema, rato fetal Ter119 fígado (marcador de células eritróides maduras) eritroblastos negativos foram purificados e transduzidas com expressão exógena de cDNAs ou pequenas hairpin RNAs (shRNAs) para os genes de interesse. As células foram posteriormente cultivadas em outras que a eritropoietina (Epo) para manter a sua fase progenitoras, durante 12 horas, permitindo que os cDNAs exógenos ou shRNAs um meio contendo factores de crescimentopara expressar. As células foram mudadas para meio de EPO depois de 12 horas para induzir a diferenciação e proliferação celular, enquanto os materiais genéticos exógenos, já foram expressos. Este protocolo facilita a análise de funções de genes na fase inicial da eritropoiese terminal. Para estudar tarde fase terminal eritropoiese, as células foram cultivadas em meio imediatamente após a transdução de Epo. Desta forma, as células já foram diferenciadas para a fase tardia da eritropoiese terminal quando os materiais genéticos transduzidas foram expressos. Recomendamos a aplicação geral desta estratégia que ajudaria a entender as funções dos genes detalhadas em diferentes estágios de eritropoiese terminal.

A Figura 1 descreve as estratégias experimentais. O protocolo consiste em duas condições independentes para alvejar as funções das moléculas sinalizadoras nas fases precoce e tardia da eritropoiese terminal. Ter119 eritroblastos de fígado fetal negativos foram purificados a partir do rato E13.5 feto. A análise por citometria de fluxo de células eritróides fetais de fígado antes e após purificação demonstrado que a purificação foi eficiente (Figuras 2A e 2B).</str...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Nós apresentamos um rato fetal sistema de cultura in vitro de eritroblastos fígado que disseca os estágios iniciais e tardios da eritropoiese terminal. Este sistema facilita a análise funcional de genes específicos em diferentes estágios de desenvolvimento.

Rato Fetal Liver Sistema de Cultura de dissecar Funções gene alvo nas fases precoces e tardias da Terminal Eritropoiese

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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Research

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Cell Biology

Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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14.8K visualizações.  Northwestern University.  Nós apresentamos um rato fetal sistema de cultura in vitro de eritroblastos fígado que disseca os estágios iniciais e tardios da eritropoiese terminal. Este sistema facilita a análise funcional de genes específicos em diferentes estágios de desenvolvimento. 

Vídeo: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Imunologia e Infecção

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Utilizando o rato como um BLT humanizado baseado Stem Modelo Gene Therapy célula tumoral

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Abordagem de processamento paralelo multi-alvo para Gene-a-estrutura Determinação da polimerase da gripe PB2 Subunidade

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Repórter Vaccinia vírus para quantificar Função Viral em todas as fases da Expressão Gênica

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Isolamento de Leucócitos a partir de tecidos de murino na interface materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Culturas placentária e Decidual órgão humano para estudar Infecções na interface materno-fetal

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Ensaio de citometria de fluxo baseada para a monitorização das funções das células NK

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Geração de variantes de fuga de neutralizar os anticorpos monoclonais de vírus da gripe

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Um método para avaliar funções efetoras mediada por Fc induzidas por anticorpos específicos de hemaglutinina de gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...