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

Den pMSCV-AID-I-Thy1.1 och pKat2 vektorer var en slags gåva från GD Schatz och pVSV-G, pRSV-Rev, och pMDLg / pRRE vektorer var en slags gåva från BR Cullen.

1. Förbereda prover och celler <ol><li> Utför en medelstor skala preparation av DNA (midiprep) av överuttryck eller shRNA innehåller konstruerar och retrovirus förpackningen vektor pKat2 eller Lentiviral vektorer förpackningen pVSV-G, pMDLg / pRRE och pRSV-Rev 25. Använd 6 mikrogram DNA för varje konstruera och antingen 4 mikrogram pKat2 (för retrovirus infektioner) eller 1,5 mikrogram DNA för varje pVSV-g, pMDLg / pRRE och pRSV-Rev vektorer förpackningar (för Lentiviral infektioner). </li><li> ...

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Som diskuterats tidigare har cellinje modeller för antikropp diversifiering blivit en populär utgångspunkt för att identifiera nya proteiner som påverkar olika steg under antikropp diversifiering. Vi presenterar här en metod för att använda virusinfektion för att antingen slå ner eller överuttrycker proteiner i Ramos B-cell linje och sedan undersöka effekterna på SHM. För dessa studier använder vi både WT Ramos och HJÄLPEN hej Ramos celler. WT celler kan användas...

Föreslagna reagenser - de flesta av dessa kan ersättas med liknande produkter från andra leverantörer. <table border="1" ><tbody><tr><th> Reagensets namn </th><th > Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td > 6-väl klart TC-behandlade plattor </td><td > Corning </td><td> 3516 </td><td ></td></tr><tr><td > 10 ml BD Luer-Loksyringes </td><td > BD Medical </td><td > 309.604 </td><td ></td></tr><tr><td> 24-väl klart TC-behandlade plattor </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > 96-väl klar plan botten polystyren TC-behandlad mikroplattor </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > 100 mm TC-behandlad kultur rätter </td><td > Corning </td><td > 430.167 </td><td ></td></tr><tr><td > Acrodisc spruta filter, 0,45 ìm </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 > Agarosen </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ampicillin </td><td> Sigma </td><td > A0166 </td><td> 100 mg / ml i vatten </td></tr><tr><td > BD FACSCanto II flödescytometer </td><td> BD Biosciences </td><td ></td><td > eller liknande </td></tr><tr><td> BD Falcon rund botten polystyren rör </td><td> BD Biosciences </td><td > 352.054 </td><td > för FACS </td></tr><tr><td > Bosc 23 celler </td><td > ATCC </td><td > CRL-11.270 </td><td ></td></tr><tr><td > CO 2-inkubator kan 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco ändrade Eagles medium) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (fetalt bovinserum) </td><td > Gemini bioprodukter </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-råtta CD90/mouse CD90.1 antikropp </td><td > BioLegend </td><td > 202.503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > FuGENE 6 Transfektion Reagens </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES buffertlösning </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA-polymeras </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > LB buljong (lysogeny buljong - Luria) Pulver </td><td > Difco </td><td > 240.230 </td><td ></td></tr><tr><td > UPPDRAG TRC shRNA bakteriell glycerol lager </td><td > Sigma </td><td ></td><td> shRNA vektorer </td></tr><tr><td > NCS (nyfödd kalv serum) </td><td > Gemini bioprodukter </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (fosfatbuffrad lösning) </td><td > Invitrogen </td><td> 70.011 </td><td > utspädd till 1x i vatten </td></tr><tr><td > PE α-humana IgM-antikroppar </td><td > BioLegend </td><td > 314.508 </td><td ></td></tr><tr><td > PGS (penicillin-streptomycin-glutamin lösning) </td><td > Gemini Bio-Paror </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine bromid) </td><td > Sigma </td><td > 107.689 </td><td > 10 mg / ml i vatten </td></tr><tr><td > PureYield Plasmid Midiprep System </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromycin </td><td> Sigma </td><td > P8833 </td><td > 250 mikrogram / ml i vatten </td></tr><tr><td > QIAquick gel utvinning kit </td><td > QIAGEN </td><td > 28.706 </td><td ></td></tr><tr><td> Ramos (RA 1) celler </td><td > ATCC </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 medelstora </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > Upphöjd II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST qPCR kit </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA-polymeras </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > Friluftskartan TA Kloning kit </td><td > Vitrogen </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Guiden SV Genomiskt DNA reningssystem </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-bromo-4-klor-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > 40 mg / ml i DMSO </td></tr></tbody></table>

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

B-celler börja sitt liv med låg affinitet antikroppar genereras av V (D) J rekombination. Men efter att upptäcka en patogen är den rörliga (V) region i ett immunglobulin (IG) genen muterad ca 100 tusen gånger mer än resten av genomet genom somatisk hypermutation (SHM), vilket resulterar i hög affinitet antikroppar 1,2. Dessutom producerar klass växla rekombination (CSR) antikroppar med olika effektorfunktioner beroende på vilken typ av immunsvar som behövs för en viss patogen. Både CSR och SHM initieras av aktivering-inducerad cytidindeaminas (AID), som deaminates cytosin rester i DNA för att producera uracils. Dessa uracils behandlas av felbenägen former av reparationer vägar, till slut leder till mutationer och rekombination 1-3. Vår nuvarande förståelse av de molekylära detaljerna i SHM och CSR kommer från en kombination av studier på möss, galvaniska element, cellinjer, och cell-free experiment. Musmodeller kvar guldmyntfoten med genetiska knockouts visar kritiska roller för många reparation faktorer (t.ex. Ung, MSH2, Msh6, Exo1 och polymeras η) 4-10. Men inte alla gener kan bli föremål för knockout studier. Till exempel knockouts av flera dubbel-strand proteiner paus reparationer embryonically dödliga eller försämra B-cells utveckling 11-14. Dessutom ibland särskilda funktion av ett protein i SHM, eller CSR kan maskeras av mer globala fel orsakade av knockout. Dessutom kan eftersom experiment på möss vara långa, ändra uttrycket av enskilda gener i cellinjer har blivit ett allt populärare första steget att identifiera och karakterisera gener 15-18. Ramos - en Burkitt lymfom cellinje som konstitutivt genomgår SHM - har varit ett populärt cell-line modell för att studera SHM 18-24. En fördel med Ramos celler är att de har en inbyggd bekväm semi-Kvantitativa mått på SHM. Vildtyp celler uttrycka IgM och, som de plockar upp mutationer, några av de mutationer som slår ut IgM uttryck. Därför testmetoder IgM förlust av fluorescens-aktiverade celler skanning (FACS) ger en snabb läs-out för nivån på SHM. En mer kvantitativ mätning av SHM kan erhållas genom att direkt sekvensering antikroppen gener. Eftersom Ramos celler är svåra att transfektera, vi producerar stabila derivat som har ökat eller sänkt uttryck av en enskild gen genom att infektera celler med retrovirus eller Lentiviral konstruktioner som innehåller antingen ett överuttryck kassett eller en kort hårnål RNA (shRNA), respektive. Här beskriver vi hur vi infektera Ramos celler och sedan använda dessa celler för att undersöka betydelsen av specifika gener på SHM (Figur 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

Vi beskriver hur du utför retrovirala eller Lentiviral infektioner i överuttryck eller shRNA innehåller konstruktioner i människans Ramos B-cellinje och hur man mäter somatiska hypermutation i dessa celler.

Bedöma Somatisk Hypermutation i Ramos B-celler efter överuttryck eller Knockdown av specifika gener

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

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