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 og pKat2 vektorer var en slags gave fra GD Schatz og pVSV-G, pRSV-Rev, og pMDLg / pRRE vektorer var en slags gave fra BR Cullen.

1. Forberedelse af prøver og celler <ol><li> Udfør en medium skala forberedelse af DNA (midiprep) af overekspression eller shRNA-holdige konstruerer og retroviral emballagen vektor pKat2 eller lentiviral emballagen vektorer pVSV-G, pMDLg / pRRE, og pRSV-Rev 25. Bruge 6 mikrogram DNA for hver konstruere og enten 4 mg af pKat2 (for retrovirale infektioner) eller 1,5 mg DNA for hver af de pVSV-g, pMDLg / pRRE, og pRSV-Rev emballage vektorer (for lentiviral infektioner). </li><li> Forbered BOSC 23 medier. For...

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Som nævnt tidligere, har cellelinje modeller for antistof diversificering blevet et populært udgangspunkt for at identificere nye proteiner, der påvirker forskellige trin i antistof diversificering. Vi præsenterer her en metode til at bruge viral infektion til enten knock-down eller overekspression proteiner i Ramos B-celle linje og derefter undersøge indvirkningen på SHM. Til disse undersøgelser anvender vi både WT Ramos og AID hi Ramos celler. WT celler kan bruges til at...

Foreslåede reagenser - de fleste af disse kan erstattes med lignende produkter fra andre leverandører. <table border="1" ><tbody><tr><th> Navn på reagenset </th><th > Firma </th><th> Katalog nummer </th><th> Kommentarer </th></tr><tr><td > 6-godt klar TC-behandlede plader </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-godt klar TC-behandlede plader </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > 96-godt klare flad bund polystyren TC-behandlet mikroplader </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > 100 mm TC-behandlede kultur retter </td><td > Corning </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc sprøjte filtre, 0,45 μm </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 > Agarose </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 vand </td></tr><tr><td > BD FACSCanto II flowcytometer </td><td> BD Biosciences </td><td ></td><td > eller lignende </td></tr><tr><td> BD Falcon rund bund polystyren rør </td><td> BD Biosciences </td><td > 352054 </td><td > til FACS </td></tr><tr><td > BOSC 23 celler </td><td > ATCC </td><td > CRL-11270 </td><td ></td></tr><tr><td > CO 2 inkubator i stand til 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco ændrede Eagle er medium) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (føtalt bovint serum) </td><td > Gemini Bio-Products </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-rotte CD90/mouse CD90.1 antistof </td><td > BioLegend </td><td > 202503 </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 bufferopløsning </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA-polymerase </td><td > KAPA Biosystemer </td><td > KK2101 </td><td ></td></tr><tr><td > LB-bouillon (lysogeny bouillon - Luria) Powder </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > MISSION TRC shRNA bakteriel glycerol lager </td><td > Sigma </td><td ></td><td> shRNA vektorer </td></tr><tr><td > NCS (nyfødte kalveserum) </td><td > Gemini Bio-Products </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (fosfatbuffer opløsning) </td><td > Invitrogen </td><td> 70011 </td><td > fortyndes til 1x i vand </td></tr><tr><td > PE α-human IgM antistof </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (penicillin-streptomycin-glutamin løsning) </td><td > Gemini Bio-Produkter </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine bromid) </td><td > Sigma </td><td > 107689 </td><td > 10 mg / ml i vand </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 mg / ml i vand </td></tr><tr><td > QIAquick gel udvinding kit </td><td > Qiagen </td><td > 28706 </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 medium </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > Hævet II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST qPCR kit </td><td > KAPA Biosystemer </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA-polymerase </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO TA Kloning kit </td><td > In 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 Genomisk DNA oprensning systemet </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-bromo-4-chlor-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 starter deres liv med lav affinitet antistoffer genereret af V (D) J rekombination. Men efter afsløring af et patogen, er den variable (V) del af et immunglobulin (Ig) genet muteret omkring 100.000 gange mere end resten af genomet ved hjælp af somatiske hypermutation (SHM), hvilket resulterer i høj affinitet antistoffer 1,2. Derudover producerer klasse skifte rekombination (CSR) antistoffer med forskellige effektor funktioner, afhængigt af, hvilken type immunrespons, der er nødvendig for en bestemt patogen. Både CSR og SHM er initieret af aktivering-induceret cytidindeaminase (AID), som deaminates cytosin rester i DNA til at producere uracils. Disse uracils behandles af fejlbehæftet former for reparation af veje, i sidste ende fører til mutationer og rekombination 1-3. Vores nuværende forståelse for de molekylære detaljer om SHM og CSR kommer fra en kombination af studier i mus, primærelementer, cellelinjer, og celle-fREE eksperimenter. Musemodeller er fortsat guldstandarden med genetiske knockouts viser kritiske roller for mange reparationer faktorer (f.eks Ung, Msh2, Msh6, Exo1, og polymerase η) 4-10. Men ikke alle gener er modtagelig for knockout undersøgelser. For eksempel er knockouts af flere dobbelt-strenget pause reparation proteiner embryonically dødelige eller forringe B-celle udvikling 11-14. Hertil kommer, nogle gange den særlige funktion af et protein i SHM eller CSR kan maskeres ved mere global fejl forårsaget af knockout. Desuden kan da eksperimenter med mus være lang, ændre udtryk for enkelte gener i cellelinier er blevet en stadig mere populær første skridt til at identificere og karakterisere kandidatgener 15-18. Ramos - en Burkitts lymfom cellelinie, der konstitutivt gennemgår SHM - har været et populært celle-line model til at studere SHM 18-24. En fordel ved Ramos celler er, at de har en indbygget i praktiske semi-Kvantitativ måling af SHM. Vildtype celler udtrykker IgM, og da de afhente mutationer, nogle af de mutationer knock out IgM udtryk. Derfor analysering IgM tab ved fluorescens-aktiveret celle scanning (FACS) giver en hurtig udlæsning for niveauet af SHM. En mere kvantitativ måling af SHM kan fås ved direkte sekventering antistoffet gener. Siden Ramos celler er vanskelige at transfect, vi producerer stabile produkter, der har øget eller sænket udtryk for en individuel gen ved at inficere celler med retroviral eller lentiviral konstruktioner, der indeholder enten en overekspression af kassette eller en kort hårnål RNA (shRNA), hhv. Her beskriver vi, hvordan vi inficere Ramos celler og derefter bruge disse celler til at undersøge betydningen af ​​specifikke 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, hvordan du udfører retroviral eller lentiviral infektioner af overekspression eller shRNA-holdige konstruerer i den menneskelige Ramos B-celle linie og hvordan man kan måle somatiske hypermutation i disse celler.

Vurdering af Somatisk Hypermutation i Ramos B-celler efter Overekspression eller Knockdown af specifikke 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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