小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测
Summary
在这里，我们提出了一个协议，利用免疫荧光染色和DNA点印法检测细胞和脑组织中5-羟基甲基细胞氨酸。
Abstract
在哺乳动物基因组中已经发现了多个DNA修饰。其中，对5-甲基细胞氨酸和5-羟基甲基细胞氨酸介导表观遗传机制进行了深入的研究。5-羟基甲基细胞氨酸在大脑胚胎发育和产后发育过程中表现出动态特征，在基因表达中起着调节作用，并涉及多种神经系统疾病。在这里，我们描述了详细的方法，包括免疫荧光染色和DNA点缀，以检测培养细胞和小鼠脑组织中5-羟基甲基细胞氨酸。
Introduction
表观遗传修饰，包括DNA修饰，组蛋白修饰和RNA修饰，已被证明在不同的生物过程和疾病1，2，3， 4,5,6,7.长期以来，DNA甲基化（即5-甲基细胞氨酸（5-mC））一直被视为高度稳定的表观遗传标记，无法在基因组中进一步修饰。最近，发现TET（十-十一位）家族蛋白（TET1、TET2和TET38、9）可氧化至5-羟基甲基细胞氨酸（5-hmC）。进一步研究表明，5-hmC可以作为一个稳定的标记，并通过调节基因表达4，10，11，12发挥生物作用。
目前的证据表明，5-hmC在神经元组织/细胞中相对于哺乳动物的其他类型的组织高度丰富，并在神经元发育13、14过程中表现出动态特征。在神经元系统中，5-hmC介导表观遗传修饰在调节神经干细胞、神经元活动、学习和记忆方面起着重要作用，并涉及多种神经系统疾病，包括雷特综合征、自闭症、阿尔茨海默氏症疾病，亨廷顿舞蹈症等2，13，15，16，17，18，19，20。
有几种方法可以检测细胞和组织中的5-hmC14、21、22、23、24。在这里，我们描述了两种方法来检测5-hmC的存在，并量化5-hmC的全球水平：免疫荧光染色和DNA点斑。这两种方法方便而敏感，在以前的研究中已成功地使用了25、26、27、28、29、30。这两种方法的关键步骤是DNA变性。对于 5-hmC 的免疫荧光染色，需要对具有 1 M HCl 的样品进行预处理。对于 5-hmC 点斑点，使用 NaOH 溶液执行 DNA 变性。这两种方法以及下一代测序是研究5-hmC功能的非常有用的工具。
Protocol
所有动物程序均已通过浙江大学动物伦理委员会批准。 1. 成人神经干细胞和神经元的培养 分离成人神经干细胞从成人（8-10周大）C57/BL6雄性小鼠前脑，如前31，32所述。 在DMEM/F-12培养基培养的成人神经干细胞中含有20纳克/mL FGF-2、20纳克/mL EGF、27补充剂、1%抗生素抗霉菌和2mM L-谷氨酰胺，在37°C的5%CO2培养箱中。</…
Representative Results
为了揭示成年小鼠海马区5-hmC的分布，我们用抗体对神经元细胞（NeuN）和5-hmC进行了免疫荧光。在海马区，5-hmC与神经元细胞标记NeuN（图1A-H）共同本地化良好，表明神经元中5-hmC的富集。 为了确定神经元发育过程中的5-hmC动力学，首先使用从增殖和分化的成人神经干细胞（NSCs）中分离出的DNA样本进行点斑点。点斑点结果显示，在NSC分化期间?…
Discussion
表观遗传修饰在大脑发育、成熟和功能中起着至关重要的作用。作为DNA修饰的稳定标记，动态5-hmC对行为适应、神经元活动有反应，与基因表达呈正相关;因此，它参与大脑的正常功能和神经紊乱4。为了探索它在细胞和组织中的功能，有必要检测5-hmC的存在，并比较治疗前后的水平。在这里，我们演示了两种检测细胞和组织5-hmC的便捷方法，这些方法可以在实验室中使用普通设备?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
XL部分得到了中国国家重点研发项目（2017YFE0196600）和国家自然科学基金（授权号31771395，31571518）的支持。Q.S.得到了国家重点研发项目（2017YFC1001703）和浙江省重点研发项目（2017C03009）的支持。W.X.得到了浙江省自然科学基金（LY18H020002）和浙江省科技厅（2017C37057）的支持。
Materials
| 4'-6-diamidino-2-phenylindole (DAPI ) | Sigma-Aldrich | D8417 |
| Adobe Photoshop software | Adobe Inc. | / |
| Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher | A11008 |
| Alexa Fluor 568 goat anti-mouse IgG | Thermo Fisher | A11001 |
| anti-5-hydroxymethylcytosine | Active Motif | 39769 |
| anti-NeuN | Millipore | MAB377 |
| B27 supplement | Gibco | 12587-010 |
| B27 supplement | Gibco | 12580-010 |
| B27 supplement | Gibco | 17504-044 |
| Cryostat microtome | Leica | CM1950 |
| DMEM/F-12 medium | OmegaScientific | DM25 |
| epidermal growth factor | PeproTech | 100-15 |
| Fibroblast growth factor-basic | PeproTech | 100-18B |
| forskolin | Sigma-Aldrich | F6886 |
| GlutaMax | Thermo | 35050061 |
| L-Glutamine | Gibco | 25030-149 |
| neurobasal medium | Gibco | 21103-049 |
| normal goat serum | Vector Laboratories | Z0325 |
| nylon membrane (Hybond™-N+ ) | Amersham Biosciences | RPN303B |
| OCT | Leica | 14020108926 |
| Pen Strep | Gibco | 15140-122 |
| phenol: chloroform: isoamyl alcohol (25: 24:1 ) | Sigma-Aldrich | 516726 |
| Poly-D-Lysine | Sigma | P0899-10 |
| proteinase K | VVR | 39450-01-6 |
| retinoic acid | Sigma-Aldrich | R2625 |
| Triton X-100 | Solarbio | T8210 |
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Citazione di questo articolo
Zhuang, Y., Chen, J., Xu, W., Shu, Q., Li, X. The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice. J. Vis. Exp. (151), e59950, doi:10.3791/59950 (2019).