利用小鼠脑肿瘤球的原发染色质沉淀
Summary
后生机制在胶质瘤中经常被改变。染色质沉淀可用于研究基因改变的后果, 胶质瘤的改变导致的组蛋白修饰, 调节染色质结构和基因转录。本协议描述了小鼠脑肿瘤球的原发染色质沉淀。
Abstract
后生修饰可能参与胶质瘤的发展和进展。癌基因和肿瘤抑制因子的促发和调控区的甲基化和再乙酰性的改变, 可能会导致在脑肿瘤的发病机制中的表达改变, 并发挥重要作用。原生染色质沉淀 (芯片) 是一种流行的技术, 允许检测的修改或其他蛋白质紧紧绑定到 DNA。与交联芯片不同的是, 在原生芯片中, 细胞不能用甲醛来将蛋白质与 DNA 进行共价键连接。这是有利的, 因为有时交联可能修复的蛋白质, 只有短暂的互动与 DNA, 并没有功能意义的基因调控。此外, 抗体通常是针对不固定的多肽。因此, 原片中抗体特异性增加。然而, 重要的是要记住, 本机芯片只适用于研究组蛋白或其他与 DNA 紧密结合的蛋白质。本协议描述了鼠脑肿瘤球的原染色质沉淀。
Introduction
后生事件在胶质瘤中很常见, 可能在肿瘤发病中起重要作用。的确, 在儿科高级别胶质瘤中, 基因组蛋白变体 H3.3 和 H3.1 编码的突变通常发生在1。突变影响组蛋白修饰, 并有主要的后生后果2,3。在青春期到成人频谱, 复发突变的异脱氢酶基因 1/2 (IDH1/2), 一种突变, 抑制α公斤依赖组蛋白和 DNA de methylasaes, 以及基因改变的其他染色质调节器, 如 ATRX 和 DAXX 发生4. 因此, 研究影响后生调节因子的突变是如何改变染色质结构和调节组蛋白修饰的至关重要的, 而这反过来又会对肿瘤细胞的转录产生巨大的影响。
染色质沉淀 (芯片) 是一种功能强大的工具, 用于评估在基因组5,6,7中表观遗传修饰的影响。在原生芯片中, 染色质是用 micrococcal 核酸 (MNase) 消化的, immunoprecipitated 使用对感兴趣的蛋白质提出的抗体, 然后从 immunoprecipitated 染色质复合体6中纯化出 DNA。细胞在过程中没有固定, 因此这种技术只适用于与 DNA6紧密交互的蛋白质的研究。缺乏交叉链接艾滋病抗体特异性, 因为抗体通常是针对不固定的多肽或蛋白质7。此外, 由于没有交叉链接的步骤, 这就减少了固定非特定的瞬时蛋白质-DNA 相互作用的几率, 而不是规范的7,8。芯片可以用来识别在特定的基因组区域中蛋白修饰的丰富性。在这里, 我们详细介绍了一个在球 (NS) 中由基因改造的小鼠胶质瘤模型来执行本机芯片的协议。
Protocol
Representative Results
Discussion
这里提供的协议将使用户能够在来自基因工程脑肿瘤的 NS 上执行本机芯片。与交叉连接芯片相比, 这个协议是有限的研究与 DNA 紧密关联的蛋白质6。所使用的单元格数可以根据需要进行修改, 并且可以扩展协议。我们每 IP 使用 1 X 106单元, 但是, 本机芯片也可以执行, 只要有 4 X 104单元格5。在本协议中, 我们通过 qPCR 分析芯片 DNA;然而, 该协议也可?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国立卫生研究院/国家神经疾病 & 中风研究所 (NIH/一) 的资助, R37-NS094804、R01-NS074387、R21-NS091555 M.G.C.;NIH/一授予 R01-NS076991、R01-NS082311 和 R01-NS096756 P.R.L.;NIH/一 R01-EB022563;神经外科部;利亚快乐的心, 和乍得虽然基金会 M.G.C. 和 P.R.L. RNA 生物医学补助金 F046166 M.G.C. F.M.M. 是支持 F31 NIH/NINDS-F31NS103500。R.I.Z.-五。由 NIH/研究院资助5T34GM007821-37。
Materials
| Agilent 2100 Bioanalyzer | Agilent | G2946-90004 | bioanalyzer |
| AccuSpin Micro 17R, refrigerated | Fisher Scientific | 13-100-676 | |
| C57BL/6 | Taconic | B6-f | C57BL/6 mouse |
| Calcium Chloride | Aldrich | 22350-6 | buffer reagent |
| D-Luciferin, Potassium Salt | Goldbio | LuckK-1g | |
| DiaMag1.5 magnetic rack | Diagenode | B04000003 | for magnetic bead washes |
| DiaMag Rotator EU | Diagenode | B05000001 | rotator |
| DMEM/F-12 | Gibco | 11330-057 | NSC component |
| Dynabeads Protein A | Thermo Fisher Scientific | 10001D | protein A magnetic beads |
| Dynabeads Protein G | Thermo Fisher Scientific | 10003D | protein G magnetic beads |
| EGF | PeproTech | AF-100-15 | prepare 20 μg/mL stock in 0.1% BSA and aliquot. |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma | E-4884 | buffer reagent |
| ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA) | Sigma | E-4378 | buffer reagent |
| Fast SYBR Green Master Mix | Applied Biosystems | 4385612 | qPCR reagent |
| Fetal Bovine Serum | Gibco | 10437028 | for freezing cells |
| FGF | PeproTech | 100-18b | Prepare 20 μg/mL stock in 0.1% BSA and aliquot. |
| Forceps | Fine Science Tools | 11008-13 | for dissection of tumor |
| Glycerol, MB Grade | EMD- Millipore | 356352 | |
| H3K4me3 | Abcam | Ab8580 | |
| H3K27me3 | Millipore | 07-449 | |
| HBSS | Gibco | 14175-103 | balanced salt solution |
| Fluriso | VETone | 501017 | inhalation anesthetic |
| Ivis Spectrum | Perkin-Elmer | 124262 | in vivo optical imaging system |
| Hyqtase | HyClon | SV3003001 | cell detachment media |
| Lithium Chloride | Sigma | L8895 | buffer reagent |
| Low binding microtubes | Corning Costar | CLS3207 | low protein binding microcentrifuge tube |
| Microcentrifuge tube | Fisher | 21-402-903 | regular microcentrifuge tube |
| Micrococcal Nuclease | Thermo Fisher Scientific, Affymetrix | 70196Y | each batch may differ; purchase sufficient amount for experiments and aliquot. |
| N2 | Gibco | 17502-048 | NSC component |
| Normal Rabbit IgG | Millipore | 12-372 | |
| Normocin | Invivogen | NOL-36-063 | anti-microbial agent, use at 0.1 mg/mL. |
| NP-40 (Igepal CA-630) | Sigma | 18896-50ML | buffer reagent |
| Kimble Kontes Pellet Pestle | Fisher Scientific | K749515-0000 | |
| Protease Inhibitor Cocktail | Sigma-Aldrich | P8340 | aliquot and store at -20 °C. |
| Protinase K | Sigma-Aldrich | P2308 | make 10 mg/mL stock in water; aliquot and store at -20 °C. |
| QIAquick PCR Purification Kit | Qiagen | 28104 | DNA purification kit |
| Scalpel | Fine Science Tools | 10007-16 | for dissection of tumor |
| Sodium Chloride | VWR | 0241-5KG | buffer reagent |
| Sodium Deoxycholate | Sigma-Aldrich | D670-25G | buffer reagent |
| Sodium Dodecyl sulfate (SDS) | Sigma | L-4390 | buffer reagent |
| Tris Base | Thermo Fisher Scientific | Bp152-1 | buffer reagent |
| Triton X-100 | Thermo Fisher Scientific | BP 151-500 | polyethylene glycol octylphenyl ether |
| Standard Mini Centrifuge | Fisherbrand | 12-006-901 | standard mini centrifuge |
| SZX16 microscope | Olympus | SZX16 | flourescent dissecting microscope |
| ViiA 7 Real-Time PCR System with Fast 96-Well Block | Applied Biosystems | 4453535 | |
| Nanodrop One | Thermo-Fisher Scientific | ND-ONEC-W |
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