评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变
Summary
我们描述了如何执行逆转录病毒或慢病毒感染的过度表达或shRNA包含人类拉莫斯的B -细胞系构造和如何衡量在这些细胞中的体细胞超突变。
Abstract
由V（D）J重组产生的低亲和力抗体的B细胞开始他们的生活。然而，检测的病原体后，变量（五）地区的免疫球蛋白（Ig）基因突变约100,000倍以上的基因组的其余通过体细胞超突变（SHM），高亲和力的抗体 1,2 。此外，类开关重组（CSR）产生一种免疫反应，是需要一个特定的病原体不同的效应功能的抗体。 CSR和SHM发起激活诱导的胞苷脱氨酶（AID），deaminates在DNA胞嘧啶残基产生uracils。这些uracils都容易出错的修复途径形式处理，最终导致突变和重组1-3。
我们目前所了解的SHM和CSR的分子细节来自相结合的研究在小鼠原代细胞，细胞系，细胞- F稀土元素的实验。小鼠模型仍显示许多修复因子（如雄，MSH2，MSH6，Exo1，和聚合酶η）4-10的关键作用的基因敲除的黄金标准。然而，并非所有的基因都为淘汰赛研究适合。例如，几双链断裂修复蛋白敲除embryonically致命或损害B细胞发展11-14。此外，有时在SHM或企业社会责任的一种蛋白的具体功能可能掩盖淘汰赛造成全球的缺陷。此外，由于在小鼠实验中，可以长篇大论，改变细胞系中的单个基因的表达已经成为一种越来越受欢迎的第一步，鉴定和表征候选基因15-18。
拉莫斯- Burkitt淋巴瘤细胞系组成进行SHM -曾流行一种细胞系模型来研究SHM 18-24。拉莫斯细胞的一个优势是，他们有一个内置的方便半定量测量的SHM。野生型细胞表达IgM和，因为他们拿起突变，一些淘汰IgM的表达的突变。因此，化验IgM的荧光激活细胞扫描仪（FACS）损失提供了SHM水平的快速读出。 SHM的定量测量，可通过直接的抗体基因测序。
由于拉莫斯细胞难以转染，我们生产的稳定增加或降低一个人的基因的表达与逆转录酶病毒或慢病毒的结构，包含一个过度表达纸盒或一个短发夹RNA（shRNA），分别感染细胞的衍生产品。在这里，我们描述了我们如何感染拉莫斯细胞，然后利用这些细胞来调查SHM特定基因的作用（图1）。
Protocol
1。准备样品和细胞执行一个中等规模制备的DNA（midiprep）的过度表达或shRNA包含结构和逆转录病毒包装载体pKat2或慢病毒包装载体pVSV – G，pMDLg / pRRE和PRSV – 冯 25 。使用每个构造中的DNA和6微克4微克pKat2（逆转录病毒感染）或每个pVSV – G，pMDLg / pRRE 1.5微克DNA，PRSV – REV包装载体（慢病毒感染）。 准备BOSC 23家媒体。转染，使用两个未经修正的贝科的改良Eagle培养基（DMEM），以及完…
Discussion
如前所述，多元化的抗体细胞株模型已经成为一种流行的出发点，以确定新的蛋白质，抗体多样化的影响在不同的步骤。我们在座的使用在拉莫斯B细胞病毒感染是击倒或过度表达的蛋白质的方法，然后检查SHM影响。
对于这些研究中，我们利用WT拉莫斯和援助您好拉莫斯细胞。野生型细胞可用于研究目标或表达的援助以及援助产生病变的修复，涉及的因素，而?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
pMSCV – AID – I – Thy1.1和pKat2载体，从DG Schatz和pVSV -摹，PRSV修订版的礼物，并pMDLg / pRRE载体是一种从BR脐周礼物。
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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Tags
Somatic HypermutationRamos B CellsOverexpressionKnockdownSpecific GenesV(D)J RecombinationAffinity AntibodiesImmunoglobulin GeneSomatic Hypermutation (SHM)Class Switch Recombination (CSR)Activation-induced Cytidine Deaminase (AID)Cytosine ResiduesUracilsRepair PathwaysMolecular DetailsMice ModelsGenetic KnockoutsRepair FactorsDouble-strand Break Repair ProteinsB-cell Development
Citer Cet Article
Upton, D. C., Unniraman, S. Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes. J. Vis. Exp. (57), e3573, doi:10.3791/3573 (2011).