Vurdering af Somatisk Hypermutation i Ramos B-celler efter Overekspression eller Knockdown af specifikke gener
Summary
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.
Abstract
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).
Protocol
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
Suggested reagents – most of these may be substituted with similar products from other vendors.
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| 6-well clear TC-treated plates | Corning | 3516 | |
| 10 mL BD Luer-Loksyringes | BD Medical | 309604 | |
| 24-well clear TC-treated plates | Corning | 3526 | |
| 96-well clear flat bottom polystyrene TC-treated microplates | Corning | 3596 | |
| 100 mm TC-treated culture dishes | Corning | 430167 | |
| Acrodisc syringe filters, 0.45 μm | Pall Life Sciences | 4604 | |
| Agar | Teknova | A7777 | |
| Agarose | GeneMate | E-3120-500 | |
| Ampicillin | Sigma | A0166 | 100 mg/mL in water |
| BD FACSCanto II flow cytometer | BD Biosciences | or similar | |
| BD Falcon round bottom polystyrene tubes | BD Biosciences | 352054 | for FACS |
| BOSC 23 cells | ATCC | CRL-11270 | |
| CO2 incubator capable of 37°C | |||
| DMEM (Dulbecco′s modified Eagle′s medium) | Sigma | D6429 | |
| FBS (fetal bovine serum) | Gemini Bio-Products | 100-106 | |
| FITC α-rat CD90/mouse CD90.1 antibody | BioLegend | 202503 | FITC α-Thy1.1 |
| FuGENE 6 Transfection Reagent | Roche | 11814443001 | |
| HEPES buffer solution | Invitrogen | 15630-080 | |
| KAPA HiFi DNA polymerase | KAPA Biosystems | KK2101 | |
| LB Broth (lysogeny broth – Luria) Powder | Difco | 240230 | |
| MISSION TRC shRNA bacterial glycerol stock | Sigma | shRNA vectors | |
| NCS (newborn calf serum) | Gemini Bio-Products | 100-504 | |
| PBS (phosphate buffered solution) | Invitrogen | 70011 | diluted to 1x in water |
| PE α-human IgM antibody | BioLegend | 314508 | |
| PGS (penicillin-streptomycin-glutamine solution) | Gemini Bio-Products | 400-110 | |
| Polybrene (hexadimethrine bromide) | Sigma | 107689 | 10 mg/mL in water |
| PureYield Plasmid Midiprep System | Promega | A2495 | |
| Puromycin | Sigma | P8833 | 250 μg/mL in water |
| QIAquick gel extraction kit | QIAGEN | 28706 | |
| Ramos (RA 1) cells | ATCC | CRL-1596 | |
| RPMI-1640 medium | Sigma | R8758 | |
| SuperScript II | Invitrogen | 18064-022 | |
| SYBR FAST qPCR kit | KAPA Biosystems | KK4601 | |
| Taq DNA Polymerase | Invitrogen | 18038-042 | |
| TOPO TA Cloning kit | Invitrogen | K4520-01 | |
| TRIzol | Invitrogen | 15596-026 | |
| Wizard SV Genomic DNA purification system | Promega | A2361 | |
| X-Gal [5-bromo-4-chloro-3-indoyl-β-D-galatopyranoside] | Growcells | C-5687 | 40 mg/mL in DMSO |
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