CK es apoyado por la Universidad de Columbia programa de postgrado. UB es miembro de la Leukemia and Lymphoma Society of America, el destinatario del Premio al Investigador de la Fundación Nueva Investigación de Leucemia y el apoyo de la Universidad de Columbia de Nueva Facultad de fondos iniciales.

1. Esplénica B-linfocitos Aislamiento y estimulación <ol><li> Cosecha del bazo de los ratones con deficiencia de AID 12 entre 2-3 meses de edad. El aislamiento del bazo se lleva a cabo en condiciones estériles y los órganos se mantienen temporalmente en solución salina fría tampón fosfato (PBS) que contiene 15% de suero fetal bovino (SFB). </li><li> El bazo es transferido a una campana de cultivo de tejidos estériles y se homogeneizaron en 5 ml de PBS suplementado con FBS al 2,5%. Transferir el homogeneizado a un tubo de 15 ml halcón. </li><li> Centrifugar la muestra homogeneizada tejido esplénico a 1000 rpm durante 10 minutos a 4 ° C para precipitar las células. </li><li> Desechar el sobrenadante tras centrifugación. Resuspender el botón celular en 10 ml de glóbulos rojos (GR) de tampón de lisis a temperatura ambiente durante 7 minutos para evitar la contaminación por los glóbulos rojos. </li><li> Centrifugar la muestra a 1000 rpm durante 10 minutos y el sobrenadante se decanta y resuspender las células en 10 ml de PBS que contenía 2,5% de SFB. </li><li> Sacar 100 l alícuota y contar las células en una dilución 1:1000 con una hemocitómetro estándar con azul tripán para excluir la presencia de células muertas. Aproximadamente 20-30 millones de células se pueden obtener por el bazo. </li><li> Centrifugar la muestra a 1000 rpm durante 10 minutos y decantar el sobrenadante. </li><li> Resuspender el precipitado en 0,5% de BSA / PBS que contenía 2 mM EDTA a una concentración de 10 7 células por 90 mL. </li><li> Añadir anti-CD43 anticuerpos magnéticas recubiertas de las células que se unen específicamente no-células B. Use 10 gotas por cada 90 l l (10 7 células). Mezclar bien e incubar a 4 ° C durante 20 minutos. </li><li> Una vez que las células han sido etiquetados, lavarlas mediante la adición de 2,5% de SFB / PBS a parte superior del tubo. Centrifugar la muestra a 1000 rpm durante 10 minutos. </li><li> Se decanta el sobrenadante. Resuspender las células en el 0,1% de BSA / PBS a una concentración de 10x10 7 células por ml. </li><li> Montar una columna de ordenación magnética en un separador magnético. Posición de un tubo cónico directamente debajo de la columna para recoger el flujo a través de la carga y la columna con 3 ml 0,5% de BSA / PBS al primer y lavado. </li><li> Una vez que el líquido de lavado ha fluido a través, reemplace con un tubo de la nueva colección. Si la muestra es menos de 1 ml, ajustar el volumen de muestra a 1 ml con 0,5% BSA / PBS / EDTA y la carga de la muestra en la columna. Cargue sólo un máximo ml de células por columna y el uso de varias columnas si es necesario. </li><li> Permitir que las células de flujo de forma pasiva a través de la columna y recoger las células no consolidados en el tubo cónico en la columna. Lavar la columna 4 veces con 3 ml de 0,5% de BSA / PBS sin dejar de recoger el flujo continuo. </li><li> Recoger todo el flujo a través de la cual las células CD43 negativo B en reposo se enriquecerá. Desechar la columna. Centrífuga de flujo continuo a 1000 rpm durante 10 minutos. </li><li> Escurrir el sobrenadante y agregar 10 ml de medio (15% de FCS / + glutamina, penicilina / estreptomicina, no aminoácidos esenciales, 50 M Β-ME) </li><li> Contar las células y la cultura a 10 6 células por ml. </li><li> Con el fin de estimular el crecimiento de las células B y la proliferación, el suplemento del medio de anticuerpos recombinantes con IL-4 y anti-CD40 de manera que la concentración final de 20 mcg / ml y 1 mg / ml, respectivamente, y la transferencia de las células de un frasco de cultivo. </li><li> Cultivar las células durante la noche. Recuento de células y diluir a 10 6 células por ml, la adición de IL-4 y CD40, según corresponda. </li><li> Células en cultivo durante 72 horas antes de la infección viral. </li></ol> 2. La transfección de células productoras Utilizar los protocolos establecidos para la transfección utilizando lípidos catiónicos o fosfato de calcio. 10 cm 3 transfectar placa por construcción de ADN, utilizando 100 mg de ADN plásmido por placa. Nosotros usamos el fosfato de calcio método de transfección 13, 14 y nota título viral alta cuando se complementa con la cloroquina. <ol><li> Use un 60% -70% confluente células como células productoras de Phoenix para ser transfectadas. En este recuento de células confluencia debe ser de 5 millones de células por placa de 10 cm. </li><li> Una hora antes de la transfección, reemplazar los medios de comunicación con nuevos medios de comunicación completa para el crecimiento celular Phoenix. </li><li> En un tubo estéril eppendorpf, se combinan 100 mg de envases construcción de ADN de 250 l de 0,5 M CaCl 2, y ajustar el volumen final de 500 ml con agua estéril </li><li> Usando una pipeta de 1 mL, mezclar enérgicamente esta solución con 500 l de la Policía Nacional 2X un tubo de polipropileno de 15 ml </li><li> Permita que esta mezcla a reposar a temperatura ambiente durante 8-10 minutos. Durante este tiempo se forma un precipitado. </li><li> Añadir la mezcla de transfección, gota a gota a las células mientras agita el medio para distribuir uniformemente la mezcla de todo. </li><li> Se incuban las células a 37 ° C durante 12 horas. </li><li> 12 horas después de la transfección, reemplazar el medio con 8 ml medio de cultivo B celular completo y volver a las células a la incubadora a 37 ° C. </li></ol> 3. La infección de las células B estimuladas esplénica <ol><li> Aspirar el sobrenadante de las células Phoenix 48 horas de grabación después de transfección y pasar el sobrenadante a través de un filtro de 0,2 micrones para eliminar los desechos celulares. </li><li> Mezclar 3 ml de virus que contienen sobrenadante con 3 ml de células B pre-activado (como en el paso 1). Con el fin de mejorar la adsorción viral a las células, agregar polibreno a una concentración final de 16 mg por ml. </li><li> Infectar a las células por centrifugación a 2500 rpm durante 90 minutos a 30 ° C. </li><li> Inmediatamente después de la vuelta, se incuban por un período de 48-72 horas a 37 ° C para permitir el crecimiento y proliferación celular. </li></ol> 4. Análisis por citometría de flujo <ol><li> El uso de un citómetro de flujo, análisis de las células de la superficie expresión de IgG1 B220 y la superficie. La presencia de IgG1 en la superficie de las células indica que la recombinación de cambio de clase se ha producido como consecuencia de rescate con éxito a través de la complementación retroviral del gen de la AID. </li></ol> 5. Resultados representante Hemos logrado rescatar la deficiencia de la proteína del factor citidina deaminasa inducida por activación (AID) de la AID-deficiente (AID-/ -) a los linfocitos B con la transducción retroviral. En pocas palabras, generamos retrovirus recombinantes que expresan AID ratón (criada) de proteínas mediante transfección de células con Phoenix PMX-criada-GFP 15-18. El virus de la cosecha fueron infectadas en las células B deficiente AID-obtenidos a partir de ratones deficientes en el AID-ex vivo recombinación de cambio de clase (CSR) condiciones de estimulación 19. Las células B infectadas fueron analizadas para que la RSE IgG1 después de 72 horas en la cultura mediante el análisis de citometría de flujo. La figura 2 muestra la detección de IgG1 en la superficie de células B con la ayuda, pero no la construcción de vacío, lo que indica que la RSE se ha producido como resultado de una ayuda de salvamento de expresión. <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="Figura 1" /> Figura 1: Esquema de envases retroviral dentro de las células de productores: gen de interés, representados por Ψ, se subclonó en PMX-IRES-GFP, como se describió previamente 15. El plásmido se transfectadas en un ecotropic línea celular de empaquetamiento viral capaz de producir gag-pol y proteína de envoltura (Phoenix, Orbigen), utilizando el método de fosfato de calcio 13, 14. Cuarenta y ocho horas después de la transfección, el sobrenadante que contiene las partículas virales se cosechó para la infección en las células diana B primarios obtenidos de los ratones. <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="Figura 2" /> Figura 2: Las células deficientes en B AID, estimularon con anti-CD40 y la IL-4 fueron infectadas con un retrovirus que contiene PMX-criada-GFP o un retrovirus que expresa GFP sola. El nivel de la RSE a la IgG1 se evaluó mediante análisis FACS.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+sequencing+and+comparative+analysis+of+the+mouse+genome.">Initial sequencing and comparative analysis of the mouse genome.</a> Nature. 420, 520-562 (2002).</li><li>Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., Lewontin, R. C., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Genetic Analysis. , (2000).</li><li>Gondo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+gene+targeting+in+the+mouse+for+functional+genomics.">Next-generation gene targeting in the mouse for functional genomics.</a> BMB Rep. 42, 315-3123 (2009).</li><li>Plachy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proviruses+selected+for+high+and+stable+expression+of+transduced+genes+accumulate+in+broadly+transcribed+genome+areas.">Proviruses selected for high and stable expression of transduced genes accumulate in broadly transcribed genome areas.</a> J Virol. 84, 4204-4211 (2010).</li><li>Felice, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+sites+are+genetic+determinants+of+retroviral+integration+in+the+human+genome.">Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.</a> PLoS One. 4, e4571-e4571 (2009).</li><li>Karavanas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+targeting+by+murine+retroviral+vectors.">Cell targeting by murine retroviral vectors.</a> Crit Rev Oncol Hematol. 28, 7-30 (1998).</li><li>Noelle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+39-kDa+protein+on+activated+helper+T+cells+binds+CD40+and+transduces+the+signal+for+cognate+activation+of+B+cells.">A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells.</a> Proc Natl Acad Sci U S A. 89, 6550-6554 (1992).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+switch+recombination+and+hypermutation+require+activation-induced+cytidine+deaminase+(AID),+a+potential+RNA+editing+enzyme.">Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.</a> Cell. 102, 553-563 (2000).</li><li>Yelle, J., Dion, M., Hamelin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+mammalian+cells+with+viral+DNA+in+optimal+culture+conditions.">Efficient transfection of mammalian cells with viral DNA in optimal culture conditions.</a> J Virol Methods. 7, 321-326 (1983).</li><li>Chen, C., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Mol Cell Biol. 7, 2745-2752 (1987).</li><li>Fagarasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+class+switching+and+differentiation+to+IgA-producing+cells+in+the+gut+lamina+propria.">In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.</a> Nature. 413, 639-6343 (2001).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AID+antibody+diversification+enzyme+is+regulated+by+protein+kinase+A+phosphorylation.">The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation.</a> Nature. 438, 508-5011 (2005).</li><li>McBride, K. 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Transducción retroviral de células B del bazo como se describe y representa en la figura 1 es un método genético que es útil en el estudio de los linfocitos B, porque muchos de los acontecimientos de desarrollo en lymphopoesis son controlados por la regulación transcripcional de 1, 2. En las últimas etapas de maduración de las células B, activación a través de CD40L es esencial para la inducción del crecimiento de las células B, la entrada en el ciclo celular y la proliferación ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>22400</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>20012</td>
<td>&nbsp;</td>
<tr>
<td>Red Blood Cell (RBC) lysis buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>CD43 beads</td>
<td>&nbsp;</td>
<td>Miltenyi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B Cell Complete Media</td>
<td>&nbsp;</td>
<td>Various components</td>
<td>Various Components</td>
<td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&mu;M &beta;-Mercaptoethanol</td>
</tr>
<tr>
<td>IL-4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD40</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>553787</td>
<td>&nbsp;</td>
<tr>
<td>polybrene</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>107689 &#x2028;</td>
<td>&nbsp;</td>
<tr>
<td>Chloroquine diphosphate salt</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>C6628</td>
<td>Used at 100mM</td>
</tr>
<tr>
<td>Phoenix Eco cells (Murine)</td>
<td>&nbsp;</td>
<td>Orbigen</td>
<td>RVC-10002</td>
<td>&nbsp;</td>
<tr>
<td>PE-Cy5-&alpha;-mouse-CD45R (B220)</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>15-0452-81</td>
<td>&nbsp;</td>
<tr>
<td>PE-&alpha;-mouse-IgG1</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>A85-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

recombinant retroviral production and infection of b cells

La expresión transgénica de genes en células eucariotas, es un poderoso método de genética inversa en la que se expresa un gen de interés bajo el control de un sistema de expresión heteróloga para facilitar el análisis del fenotipo resultante. Este enfoque puede ser usado para expresar un gen que no se encuentra normalmente en el organismo, para expresar una forma mutante de un gen producto, o sobre-expresa una forma dominante negativa del producto del gen. Es particularmente útil en el estudio del sistema hematopoyético, que la regulación transcripcional es un mecanismo de control importante en el desarrollo y diferenciación de células B 1, revisada en 02.04. La genética del ratón es una herramienta poderosa para el estudio de los genes y las enfermedades. Un análisis comparativo con el ratón y el genoma humano revela la conservación de synteny en más del 90% del genoma 5. Además, gran parte de la tecnología utilizada en modelos de ratón es aplicable al estudio de los genes humanos, por ejemplo, alteraciones de genes y la sustitución alélica 6 . Sin embargo, la creación de un ratón transgénico que requiere una gran cantidad de recursos tanto de carácter financiero y técnico. Varios proyectos han comenzado a compilar las bibliotecas de eliminar cepas de ratón (KOMP, EUCOMM, NorCOMM) o mutagénesis inducida por las cepas (RIKEN), que requieren grandes esfuerzos y la colaboración 7. Por lo tanto, es deseable que el primer estudio del fenotipo de un gen deseado en un modelo de cultivo celular de células primarias antes de pasar a un modelo de ratón. ADN retroviral se integra en el ADN del huésped, de preferencia dentro de o cerca de las unidades de transcripción o de las islas CpG, lo estable y heredable expresión del gen de empaquetado de interés y evitar el silenciamiento transcripcional 8 9. Los genes se transcriben bajo el control de un promotor de la eficiencia retroviral de alta, resultando en una alta eficiencia de la transcripción y la producción de proteínas. Por lo tanto, la expresión retroviral se puede utilizar con las células que son difíciles de transfectar, siempre y cuando las células están en un estado activo durante la mitosis. Debido a que los genes estructurales del virus se encuentran dentro de la línea celular de empaquetamiento, los vectores de expresión que se usa para clonar el gen de interés no contienen genes estructurales del virus, que no sólo elimina la posibilidad de reversiones viral y aumenta la seguridad de trabajar con los sobrenadantes virales como no viriones infecciosos se producen 10. Aquí se presenta un protocolo para la producción recombinante de retrovirales y la posterior infección de las células B del bazo. Tras el aislamiento, las células del bazo cultivadas son estimulados con linfocinas Th derivados y anti-CD40, lo que induce a una explosión de la proliferación de células B y diferenciación 11. Este protocolo es ideal para el estudio de los acontecimientos que se presentan en el desarrollo de células B y la diferenciación, las células B están aislados del bazo tras los primeros eventos hematopoyéticas, pero antes de la estimulación antigénica para inducir la diferenciación plasmáticas.

Watch this Scientific Journal Video about Recombinant Retroviral Production and Infection of B Cells at JoVE.com

Recombinant Retroviral Production and Infection of B Cells

Un sistema eficiente de la estructura y análisis de la función de un gen en un<em&gt; Ex vivo</em&gt; Cultura de los linfocitos B del bazo se describe. Este método tiene la ventaja de la producción recombinante de antirretrovirales en una línea gratuita de ayuda, ecotrófica celular de empaquetamiento. Estables, heredables expresión de un gen de interés dentro de los linfocitos primarios se logra llevando a la generación de anticuerpos de superficie de las células B sometidos a la recombinación de cambio de clase.

Recombinante de producción retrovirales y la infección de células B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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Immunology and Infection

14.0K vistas.  Columbia University College of Physicians and Surgeons. Un sistema eficiente de la estructura y análisis de la función de un gen en un Ex vivo Cultura de los linfocitos B del bazo se describe. Este método tiene la ventaja de la producción recombinante de antirretrovirales en una línea gratuita de ayuda, ecotrófica celular de empaquetamiento. Estables, heredables expresión de un gen de interés dentro de los linfocitos primarios se logra llevando a la generación de anticuerpos de superficie de las célula...

Video: Recombinant Retroviral Production and Infection of B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-activación de las células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Investigación

Inmunología e Infección

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transducción retroviral de receptores de células T en ratones las células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

Producción y Titulación de recombinantes adeno-asociado vectores virales

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Producción eficiente de parvovirus recombinante con la ayuda de derivados de adenovirus-Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Detección de True IgE que expresan las células del linaje B de ratón

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retroviral transducción de células progenitoras de médula ósea para generar las células T del receptor Retrogenic ratones

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Dirigida a tioles de la cisteína para in Vitro específica glicosilación de proteínas recombinantes

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

"Hígado-en-un-Chip" cultivos de hepatocitos primarios y células de Kupffer para infección del Virus de la Hepatitis B

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Sobreexpresión retroviral de CXCR4 en células murinas B-1a y transferencia adoptiva para la migración celular B-1a dirigida a la médula ósea y la producción de IgM

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...