Name: assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes
Uploaded: 2011-11-01T13:00:00
Description: Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Il pMSCV-AID-I-Thy1.1 e pKat2 vettori erano un gentile dono della DG Schatz e pVSV-G, pRSV-Rev, e pMDLg / pRRE vettori erano un gentile dono da BR Cullen.

1. Preparazione dei campioni e le cellule <ol><li> Eseguire una preparazione media scala del DNA (midiprep) della sovraespressione o shRNA contenenti costrutti e il vettore retrovirale imballaggio pKat2 o vettori lentivirali imballaggio pVSV-G, pMDLg / pRRE e pRSV-Rev 25. Usare 6 mg per ogni DNA costruire e uno di 4 microgrammi pKat2 (per infezioni retrovirali) o 1,5 mg di DNA per ciascuna delle pVSV-g, pMDLg / pRRE e pRSV-Ap vettori di imballaggio (per le infezioni lentivirali). </li><li> Preparare 23 BOSC...

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Come discusso precedentemente, i modelli di linee cellulari per la diversificazione degli anticorpi sono diventati un punto di partenza per identificare nuove proteine ​​che influenzano le diverse fasi durante la diversificazione degli anticorpi. Vi presentiamo qui un metodo per l&#39;utilizzo di infezione virale alle proteine ​​o knock-down o iperespressione in cellule B Ramos linea e quindi esaminare l&#39;impatto sulla SHM. Per questi studi, ci avvaliamo sia Ramos WT e AID hi...

Reagenti suggerito - la maggior parte di questi possono essere sostituiti con prodotti simili di altri vendor. <table border="1" ><tbody><tr><th> Nome del reagente </th><th > Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td > 6-ben chiaro TC-trattati piastre </td><td > Corning </td><td> 3516 </td><td ></td></tr><tr><td > 10 mL BD Luer-Loksyringes </td><td > BD Medical </td><td > 309604 </td><td ></td></tr><tr><td> 24-ben chiaro TC-trattati piastre </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > 96-polistirolo ben chiaro fondo piatto TC-trattati micropiastre </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > TC-100 millimetri trattati cultura piatti </td><td > Corning </td><td > 430167 </td><td ></td></tr><tr><td > Filtri per siringa Acrodisc, 0,45 micron </td><td > Pall Life Sciences </td><td > 4604 </td><td ></td></tr><tr><td > Agar </td><td > Teknova </td><td > A7777 </td&gt; <td ></td></tr><tr><td > Agarosio </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ampicillina </td><td> Sigma </td><td > A0166 </td><td> 100 mg / mL in acqua </td></tr><tr><td > BD FACSCanto II citometro a flusso </td><td> BD Biosciences </td><td ></td><td > o simili </td></tr><tr><td> BD Falcon rotondo fondo polistirolo tubi </td><td> BD Biosciences </td><td > 352054 </td><td > per FACS </td></tr><tr><td > BOSC 23 celle </td><td > ATCC </td><td > CRL-11270 </td><td ></td></tr><tr><td > CO 2 incubatore capace di 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (medio Dulbecco modificato Eagle) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (siero fetale bovino) </td><td > Gemini Bio-Prodotti </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-ratto CD90/mouse CD90.1 anticorpi </td><td > BioLegend </td><td > 202503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > FuGENE 6 Transfection Reagent </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES soluzione tampone </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA polimerasi </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > LB Broth (brodo lisogenia - Luria) Polvere </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > MISSIONE TRC shRNA magazzino glicerolo batterico </td><td > Sigma </td><td ></td><td> shRNA vettori </td></tr><tr><td > NCS (siero di vitello neonato) </td><td > Gemini Bio-Prodotti </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (soluzione tampone fosfato) </td><td > Invitrogen </td><td> 70011 </td><td > diluito a 1x in acqua </td></tr><tr><td > PE-α umano anticorpi IgM </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (penicillina-streptomicina-glutamina soluzione) </td><td > Gemini Bio-Prodotti </td><td > 400-110 </td><td ></td></tr><tr><td> Polibrene (hexadimethrine bromuro) </td><td > Sigma </td><td > 107689 </td><td > 10 mg / ml in acqua </td></tr><tr><td > PureYield sistema Midiprep plasmidi </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromicina </td><td> Sigma </td><td > P8833 </td><td > 250 mcg / ml in acqua </td></tr><tr><td > QIAquick gel kit di estrazione </td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td> Ramos (RA 1) le cellule </td><td > ATCC </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 medium </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > SuperScript II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR VELOCE qPCR kit </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA polimerasi </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO TA Cloning Kit </td><td > Vitrogenerazione </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Wizard SV sistema di purificazione del DNA genomico </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-bromo-4-cloro-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > 40 mg / ml in DMSO </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

Le cellule B iniziano la loro vita con gli anticorpi a bassa affinità generato da V (D) J ricombinazione. Tuttavia, al rilevamento un agente patogeno, la (V) regione variabile di un gene immunoglobuline (Ig) è mutato circa 100.000 volte più del resto del genoma attraverso la mutazione somatica (SHM), con conseguente 1,2 anticorpi ad alta affinità. Inoltre, passare ricombinazione di classe (CSR) produce anticorpi con differenti funzioni effettrici a seconda del tipo di risposta immunitaria che è necessario per un particolare agente patogeno. Sia CSR e SHM vengono avviate attraverso l&#39;attivazione indotta citidina deaminasi (AID), che deamina residui di citosina nel DNA per produrre uracils. Queste uracils sono trattati con forme soggette a errori di percorsi di riparazione, alla fine portano a mutazioni e la ricombinazione 1-3. La nostra attuale comprensione dei dettagli molecolari di SHM e RSI provenire da una combinazione di studi in topi, pile, di linee cellulari e cellule-free esperimenti. Modelli murini rimangono il gold standard con fori genetica che mostrano ruoli critici per la riparazione di molti fattori (ad esempio Ung, MSH2, MSH6, Exo1 e polimerasi η) 4-10. Tuttavia, non tutti i geni sono suscettibili per gli studi di eliminazione diretta. Per esempio, KO di proteine ​​diverse rottura a doppio filamento di riparazione sono embrionalmente letali o mettere in pericolo lo sviluppo delle cellule B 11-14. Inoltre, a volte la funzione specifica di una proteina in SHM o CSR può essere mascherato da più difetti globale causato dalla eliminazione diretta. Inoltre, dal momento che gli esperimenti sui topi possono essere lunghe, alterando l&#39;espressione di singoli geni in linee cellulari è diventata una tappa sempre più popolare primi a identificare e caratterizzare geni candidati 15-18. Ramos - una linea cellulare di linfoma di Burkitt che subisce costitutivamente SHM - è stata una popolare linea cellulare modello per studiare SHM 18-24. Un vantaggio di cellule Ramos è che hanno un built-in semi conveniente-Quantitativa misura di SHM. Cellule wild-type esprimere IgM e, come raccogliere le mutazioni, alcune delle mutazioni knock out espressione IgM. Pertanto, il saggio perdita IgM di fluorescenza attivata la scansione cellulare (FACS) fornisce una rapida lettura per il livello di SHM. Una misura più quantitativa di SHM possono essere ottenute direttamente sequenziamento dei geni anticorpali. Dato che le cellule Ramos è difficile trasfezione, produciamo derivati ​​stabili che hanno aumentato o diminuito l&#39;espressione di un singolo gene da infettare le cellule con costrutti retrovirali o lentivirali che contengono una cassetta iperespressione o un breve hairpin RNA (shRNA), rispettivamente. Qui, descriviamo il modo in cui infettare le cellule Ramos e quindi utilizzare queste cellule per studiare il ruolo di specifici geni in SHM (Figura 1).

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

Ci descrivono come eseguire infezioni retrovirali o lentivirali di sovraespressione o shRNA contenenti costruisce nell&#39;essere umano a cellule B Ramos line e come misurare mutazione somatica in queste cellule.

Valutare mutazione somatica nel Ramos cellule B dopo iperespressione o Knockdown di specifici geni

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Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...