CK è supportato dal programma Columbia University Graduate. UB è Fellow della Leukemia and Lymphoma Society of America, il destinatario del Premio New Investigator dalla Fondazione per la Ricerca leucemia ed è supportato da Facoltà Columbia University di New start-up fondi.

1. Splenica B-linfociti isolamento e stimolazione <ol><li> Raccolta della milza di topi con deficit AID 12 tra 2-3 mesi di età. L&#39;isolamento della milza viene eseguita in condizioni sterili e gli organi sono temporaneamente conservati in soluzione fisiologica fredda tampone fosfato (PBS) contenente 15% siero fetale bovino (FBS). </li><li> La milza è poi trasferito in una cappa sterile colture di tessuti e omogeneizzato in 5 ml di PBS integrata con il 2,5% FBS. Trasferire l&#39;omogeneizzato in una provetta da 15 ml falco. </li><li> Centrifugare il campione omogeneizzato di tessuto splenico a 1000rpm per 10 minuti a 4 ° C per agglomerare le cellule. </li><li> Decantare il surnatante dopo centrifugazione. Risospendere il pellet cellulare in 10 ml di globuli rossi (RBC) tampone di lisi a temperatura ambiente per 7 minuti per evitare la contaminazione da parte dei globuli rossi. </li><li> Centrifugare campione a 1000 rpm per 10 minuti e decantare il surnatante e risospendere le cellule in 10 ml di PBS contenente 2,5% FBS. </li><li> Rimuovere 100 microlitri aliquota e contare le cellule a una diluizione 1:1000 utilizzando un emocitometro standard utilizzando trypan blu per escludere qualsiasi cellule morte. Circa 20-30 milioni di cellule possono essere ottenuti per milza. </li><li> Centrifugare campione a 1000 rpm per 10 minuti e decantare il surnatante. </li><li> Risospendere il pellet in 0,5% BSA / PBS contenente 2mM EDTA a una concentrazione di 10 7 cellule per 90 microlitri. </li><li> Aggiungi anticorpi anti-CD43 sfere magnetiche rivestito le cellule di legare specificamente cellule non-B. Usa 10 perle microlitri per 90 microlitri (10 7 cellule). Mescolare bene e incubare a 4 ° C per 20 minuti. </li><li> Una volta che le cellule sono stati etichettati, lavarli con l&#39;aggiunta di 2,5% FBS / PBS alla parte superiore del tubo. Centrifugare campione a 1000 rpm per 10 minuti. </li><li> Decantare il surnatante. Risospendere le cellule in 0,1% BSA / PBS a una concentrazione di 10x10 7 celle per ml. </li><li> Montare una colonna magnetica ordinamento su un separatore magnetico. Posizionare un tubo conico direttamente sotto la colonna di raccogliere il flusso-through e caricare la colonna con 3 ml 0,5% BSA / PBS al primo e lavare. </li><li> Una volta che il liquido di lavaggio ha scorreva, sostituirlo con un tubo di nuova collezione. Se il campione è inferiore a 1 ml, regolare il volume campione ad 1 ml con 0,5% BSA / PBS / EDTA e caricare il campione nella colonna. Caricare solo un massimo di 1 ml di cellule per colonna e utilizzare più colonne, se necessario. </li><li> Permettono alle cellule di flusso passivamente attraverso la colonna e raccogliere le cellule non legato nel tubo conico sotto la colonna. Lavare la colonna 4 volte con 3 ml 0,5% BSA / PBS, pur continuando a raccogliere le flow-through. </li><li> Raccogliere tutte le flow-through, in cui le cellule CD43 negative B riposo sarà arricchita. Eliminare la colonna. Centrifuga flow-through a 1000 rpm per 10 minuti. </li><li> Scolate il surnatante e aggiungere 10 ml di mezzo (15% FCS / + glutammina, Pen / Strep, aminoacidi non essenziali, 50 mM Β-ME) </li><li> Contare le celle e la cultura a 10 6 cellule per ml. </li><li> Al fine di stimolare la crescita e la proliferazione delle cellule B, integrare il supporto con anticorpo IL-4 e anti-CD40 in modo tale che le concentrazioni finali sono 20 mg / ml e 1 mg / ml, rispettivamente, e trasferire le cellule in un pallone cultura. </li><li> Coltura delle cellule durante la notte. Contare le celle e diluire a 10 6 cellule per ml, l&#39;aggiunta di IL-4 e CD40 a seconda dei casi. </li><li> Celle di coltura per 72 ore prima dell&#39;infezione virale. </li></ol> 2. Trasfezione delle cellule Produttore Utilizzano i protocolli stabiliti per la trasfezione utilizzando lipidi cationici o fosfato di calcio. Trasfezione 10 centimetri 3 Piastra per costruire il DNA, utilizzando 100 mg di DNA plasmidico per piastra. Noi abitualmente uso di fosfato di calcio metodo di trasfezione 13, 14 e nota alto titolo virale quando è integrato con la clorochina. <ol><li> Usa il 60% -70% delle cellule confluenti Phoenix come cellule produttore per essere transfettate. A questo conta delle cellule confluenza dovrebbe essere di 5 milioni di cellule per 10 piastra cm. </li><li> Un&#39;ora prima della transfezione, sostituire il supporto con freschi multimediale completa per la crescita cellulare Phoenix. </li><li> In una provetta sterile eppendorpf, unire 100 mg di imballaggio costruire DNA, 250 ml di 0,5 M CaCl 2, e regolare il volume finale a 500 microlitri di acqua sterile </li><li> Usando una pipetta da 1 ml, mescolare vigorosamente questa soluzione con 500 microlitri HNP 2X in un tubo di polipropilene 15 ml </li><li> Lasciare il composto a riposare a temperatura ambiente per 8-10 minuti. Durante questo periodo si forma un precipitato. </li><li> Aggiungere la miscela di trasfezione goccia a goccia per le cellule, mentre vorticose del mezzo per distribuire uniformemente il composto in tutto. </li><li> Incubare le cellule a 37 ° C per 12 ore. </li><li> 12 ore dopo la trasfezione, sostituire il supporto con 8ml completo mezzo di crescita delle cellule B e le cellule ritorno ai 37 ° C incubatore. </li></ol> 3. L&#39;infezione di cellule stimolate B splenica <ol><li> Rimuovere il surnatante dalle cellule Phoenix 48 ore post-transfection e passare il sovranatante attraverso un filtro 0,2 micron per rimuovere i detriti. </li><li> Mescolare 3 mL di virus contenenti surnatante con 3 ml di pre-cellule B attivate (come al punto 1). Al fine di migliorare l&#39;adsorbimento virale alle cellule, aggiungere polibrene ad una concentrazione finale di 16 mg per ml. </li><li> Infettare le cellule mediante centrifugazione a 2500 rpm per 90 minuti a 30 ° C. </li><li> Immediatamente dopo lo spin, incubare per ulteriori 48-72 ore a 37 ° C per consentire la crescita cellulare e la proliferazione. </li></ol> 4. Citometria a Flusso Analisi <ol><li> Utilizzando un citofluorimetro, analizzare le cellule per l&#39;espressione superficiale di IgG1 B220 e superficie. La presenza di IgG1 sulla superficie delle cellule indica che l&#39;interruttore di ricombinazione di classe si è verificato a seguito di salvataggio di successo attraverso la complementazione retrovirali del gene AID. </li></ol> 5. Rappresentante Risultati Abbiamo liberato con successo la carenza del fattore proteico citidina deaminasi indotta da attivazione (AID) da AID-deficienti (AID-/ -) linfociti B con trasduzione retrovirale. In breve, abbiamo generato retrovirus ricombinante esprimente SOCCORSO mouse (cameriera) proteina trasfezione delle cellule Phoenix con PMX-Maid-GFP 15-18. I virus raccolti sono stati infettati in AID-deficienti cellule B ottenute da AID topi con deficit di ricombinazione ex interruttore classe vivo (CSR) condizioni di stimolazione 19. Le cellule B infettate sono stati poi analizzati per la RSI a IgG1 dopo 72 ore di cultura in citometria a flusso di analisi. La figura 2 mostra la rilevazione di IgG1 sulla superficie delle cellule B con aiuti, ma non il costrutto vuoto, a indicare che la RSI si è verificato a seguito di aiuti al salvataggio espressione. <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="Figura 1" /> Figura 1: Schema di imballaggio retrovirale all&#39;interno delle cellule produttore: Gene di interesse, rappresentate da Ψ, era subcloned in PMX-IRES-GFP, come descritto in precedenza 15. Il plasmide è stato transfettate in una linea cellulare di packaging ecotropic virale in grado di produrre gag-pol e proteine ​​busta (Phoenix, Orbigen), utilizzando il metodo di fosfato di calcio 13, 14. Quarantotto ore dopo la trasfezione, surnatante contenente particelle virali è stato raccolto per l&#39;infezione in cellule B target primario ottenuti da topi. <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="Figura 2" /> Figura 2: le cellule B SOCCORSO carenti, stimolati con anti-CD40 e IL-4 sono state infettate con un retrovirus contenente PMX-Maid-GFP o un retrovirus che esprime GFP solo. Il livello di CSR per IgG1 è stata valutata mediante analisi FACS.

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Nessun conflitto di interessi dichiarati.

Trasduzione retrovirale delle cellule B della milza, come descritto qui e come illustrato nella figura 1 è un approccio genetico che è utile nello studio dei linfociti B, perché molti degli eventi di sviluppo in lymphopoesis sono controllati da regolazione trascrizionale 1, 2. Negli ultimi stadi di maturazione delle cellule B, innescando via CD40L è essenziale per l&#39;induzione della crescita delle cellule B, l&#39;ingresso nel ciclo cellulare e la proliferazione 11, 20. Come...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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>
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recombinant retroviral production and infection of b cells

L&#39;espressione transgenica di geni nelle cellule eucariotiche è un potente approccio inverso genetica in cui è espresso un gene di interesse sotto il controllo di un sistema di espressione eterologo per facilitare l&#39;analisi del fenotipo risultante. Questo approccio può essere utilizzato per esprimere un gene che non si trova normalmente nell&#39;organismo, per esprimere una forma mutante di un prodotto del gene, oppure a un eccesso di esprimere un dominante negativo forma del prodotto genico. E &#39;particolarmente utile nello studio del sistema ematopoietiche, dove regolazione trascrizionale è un meccanismo di controllo importante nello sviluppo e nella differenziazione delle cellule B 1, recensione in 2-4. Genetica del topo è un potente strumento per lo studio dei geni e delle patologie umane. Un&#39;analisi comparativa del mouse e genoma umano rivela conservazione delle synteny in oltre il 90% del genoma 5. Inoltre, gran parte della tecnologia utilizzata in modelli murini è applicabile allo studio dei geni umani, per esempio, le interruzioni dei geni e la sostituzione allelica 6 . Tuttavia, la creazione di un topo transgenico richiede una grande quantità di risorse sia di natura tecnica e finanziaria. Diversi progetti hanno cominciato a compilare le librerie di ceppi di topi knock out (KOMP, EUCOMM, NorCOMM) o mutagenesi indotta da ceppi (RIKEN), che richiedono grandi sforzi e la collaborazione 7. Pertanto, è auspicabile primo studio il fenotipo di un gene desiderato in un modello di coltura cellulare di cellule primarie prima di passare a un modello murino. DNA retrovirale integra nel DNA ospite, preferibilmente all&#39;interno o in prossimità di unità di trascrizione o isole CpG, con conseguente stabile e ereditabile espressione del gene di interesse confezionato evitando silenziamento trascrizionale 8 9. I geni sono poi trascritti sotto il controllo di un promotore retrovirali ad alta efficienza, con una conseguente alta efficienza di trascrizione e produzione di proteine. Pertanto, l&#39;espressione retrovirali possono essere utilizzati con le cellule di difficile trasfezione, a condizione che le cellule sono in uno stato attivo durante la mitosi. Perché i geni strutturali del virus sono contenuti all&#39;interno della linea cellulare di packaging, i vettori di espressione utilizzata per clonare il gene di interesse non contengono geni strutturali del virus, che sia elimina la possibilità di revertanti virale e aumenta la sicurezza di lavorare con surnatanti virale come non virioni infettivi vengono prodotti 10. Qui vi presentiamo un protocollo per la produzione ricombinante retrovirali e successiva infezione delle cellule B della milza. Dopo l&#39;isolamento, le cellule coltivate splenica sono stimolati con Th linfochine derivati ​​e anti-CD40, che induce una raffica di proliferazione e differenziazione delle cellule B 11. Questo protocollo è l&#39;ideale per lo studio di eventi che si verificano in ritardo nello sviluppo delle cellule B e la differenziazione, le cellule B sono isolate dalla milza seguenti iniziale eventi ematopoietiche, ma prima di stimolazione antigenica per indurre la differenziazione plasmocitaria.

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 efficiente sistema di struttura e analisi funzionale di un gene in un<em&gt; Ex vivo</emCultura&gt; splenica di linfociti B è descritto. Questo metodo si avvale della produzione retrovirali ricombinanti in un libero helper, ecotrophic linea cellulare di packaging. Stabile, ereditari espressione di un gene di interesse all&#39;interno di linfociti primario è raggiunto porta alla generazione di anticorpi di superficie sulle cellule B in fase interruttore ricombinazione classe.

Produzione ricombinante retrovirali e infezione delle cellule 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 ...

recombinant-retroviral-production-and-infection-of-b-cells

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Immunology and Infection

14.0K Views.  Columbia University College of Physicians and Surgeons. Un efficiente sistema di struttura e analisi funzionale di un gene in un Ex vivo splenica di linfociti B è descritto. Questo metodo si avvale della produzione retrovirali ricombinanti in un libero helper, ecotrophic linea cellulare di packaging. Stabile, ereditari espressione di un gene di interesse all'interno di linfociti primario è raggiunto porta alla generazione di anticorpi di superficie sulle cellule B in fase interruttore ricombinazione...

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.

Modello murino di CD40-attivazione delle cellule B

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

Ricerca

Immunologia e infezioni

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

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule 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.

Produzione e titolazione di vettori virali ricombinanti adeno-associato

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).

Efficienti di produzione Parvovirus ricombinante con l&#39;aiuto delle Adenovirus sistemi derivati

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.

Rilevazione di True IgE-esprimono mouse B Cells Lineage

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.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

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.

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore 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 ...

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.

Targeting dei tioli cisteina per in Vitro site-specific glicosilazione delle proteine ricombinanti

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.

"Fegato-on-a-Chip" colture di epatociti primari e cellule di Kupffer per infezione da Virus dell'epatite 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.

Sovraespressione retrovirale di CXCR4 su cellule Murine B-1a e trasferimento adottivo per migrazione mirata di cellule B-1a verso il midollo osseo e la produzione di IgM

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

Methods Article

Produzione ricombinante retrovirali e infezione delle cellule B