单卵母细胞的亚硫酸氢钠诱变
Summary
亚硫酸氢钠诱变是分析DNA甲基化的黄金标准。我们修改后的协议允许在单细胞水平和DNA甲基化分析，是专门设计为个别的卵母细胞。它也可以用于卵裂阶段的胚胎。
Abstract
表观遗传学包括所有遗传和可逆的修改，染色质改变基因的辅助功能，从而调节基因转录1的主要机制。 DNA甲基化是表观遗传修饰作用主要是为镇压商标。通过共价除了到CpG二核苷酸的胞嘧啶甲基组，它可以聘请额外的镇压蛋白和组蛋白修饰，启动过程冷凝染色质和基因沉默2。 DNA甲基化是必不可少的正常发展，因为它起着关键作用，在发展规划，细胞分化，逆转录病毒分子的镇压，X染色体失活和基因组印记。
DNA甲基化分析的最有力的方法之一是亚硫酸氢钠诱变。亚硫酸氢钠是一种DNA诱变，deaminates胞嘧啶为uracils的。经过PCR扩增和seque ncing，这些转换事件被检测为胸腺嘧啶。甲基化的胞嘧啶脱氨和保护，从而继续为胞嘧啶，使单个核苷酸的3级鉴定的DNA甲基化。先进的亚硫酸氢钠诱变法的发展已经从最初报告对那些更敏感和重复性7 4-6。一个关键的进步是在琼脂糖珠嵌入少量的DNA，从而保护了苛刻的亚硫酸氢钠治疗8 DNA。这使甲基化分析，对卵母细胞和囊胚阶段的胚胎，9池进行。最先进的亚硫酸氢钠诱变协议日期是10个人囊胚阶段的胚胎。然而，由于囊胚平均64个单元（含120-720 PG基因组DNA），这个方法是不是个别的卵母细胞或卵裂期胚胎的甲基化研究有效。
帐篷“从琼脂糖嵌入包括卵母细胞11分钟的DNA金额以线索，在这里，我们提出了卵母细胞，即是直接嵌入在琼脂糖和裂解液珠紧随其后的检索和卵母细胞的透明带去除的方法，这使我们能够绕过单卵母细胞的亚硫酸氢钠诱变的两个主要挑战：保护的DNA微量降解，并在随后的众多协议的步骤的损失，更重要的是，从单一的卵母细胞获得的数据作为PCR泳池内的偏见问题被淘汰此外。 ，不慎卵丘细胞污染是用这种方法检测，可从分析12排除，因为任何一个以上的甲基化模式的样本。该协议提供了一个在单细胞水平上的成功和可重复性的DNA甲基化分析方法的改进非常适合个别的卵母细胞以及卵裂期胚胎。Protocol
第1天 GIBCO公司的水，如用无菌蒸馏水，准备当天收集的卵母细胞的新鲜以下的解决方案。为了减少DNA污染的机会，经常更换手套，并使用过滤嘴。保持管的角度离开时开放，并在不使用时，回顾一下所有的管子。我们建议的解决方案为N +1。 3％低熔点琼脂糖 30毫克低熔点（LMP）的琼脂糖 1毫升GIBCO公司H 2 O 溶于70°C间<p…
Discussion
这种单卵母细胞的检测数字包含了许多步骤，是至关重要的，需要特殊照顾。首先是卵母细胞洗涤。尤其重要的是洗每个卵母细胞的新鲜培养基中多次下降后，透明质酸酶处理以去除尽可能多的卵丘细胞。此外，卵母细胞的透明带去除酸性台氏解决方案时，要确保周围介质是清晰的卵丘细胞。卵母细胞的卵去除是非常棘手的，任何周围的卵丘细胞可以很容易地成为坚持的卵母细胞。删除一个被套?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由西安大略大学妇产科学系;支持和补助金从Ministryof研究和创新，早期的研究者奖“ER06-02-188。 MMD的支持，在的CIHR训练计划繁殖，早期发育和健康（REDIH）研究生奖学金的影响。
Materials
Table of specific reagents and equipment.
| Name of the reagent | Company | Catalogue number | Comments |
| Oocyte Collection | |||
| Hyaluronidase | Sigma | H4272 | |
| Acidic Tyrode | Sigma | T1788 | |
| Proteinase K | Sigma | P5568 | |
| 10% IGEPAL | Bioshop | NON999.500 | |
| Lysis Solution | |||
| Tris pH 7.5 | Bioshop | TRS001.5 | |
| LiCl | Sigma | L9650 | |
| EDTA pH 8.0 | Sigma | E5134 | |
| LiDS | Bioshop | LDS701.10 | |
| DTT | Invitrogen | P2325 | |
| SDS Lysis Buffer | |||
| TE pH 7.5 |
Bioshop(Tris) Sigma (EDTA) |
TRS001.5 E5134 |
|
| 10% SDS | Bioshop | SDS001.500 | |
| Bisulfite Conversion | |||
| Sodium Hydroxide | Sigma | S8045 | |
| Sodium Hydrogensulfite (Sodium Bisulfite) | Sigma | 243973 | |
| Hydroquinone | Sigma | H9003 | |
| Low Melting Point (LMP) Agarose | Sigma | A9414 | |
| Mineral Oil | Sigma | M8410 | |
| M2 Medium | Sigma | M7167 | |
| GIBCO Distilled water | Invitrogen | 15230-196 | |
| Autoclaved double distilled (dd) water | |||
| PCR | |||
| Illustra Hot Start Mix RTG | GE Healthcare | 28-9006-54 | |
| 240 ng/ml yeast tRNA | Invitrogen | 15401-011 | |
| 5x Green GoTaq Reaction Buffer | Promega | M7911 | |
| Inner and outer nested primers | Sigma | ||
| Ligation | |||
| Promega pGEM-T Easy Vector | Fisher Scientific | A1360 | |
| TA Cloning | |||
| Competent E.coli cells | Zymo Research Corp. | T3009 | |
| Equipment | |||
| Dissecting Microscope | |||
| 70°C and 90°C Heat Blocks | |||
| 37°C and 50°C Waterbaths (42°C for transformations) | |||
| Rocker | |||
| PCR machine | |||
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Tags
Single Oocyte Bisulfite MutagenesisEpigeneticsChromatin ModificationsGene AccessibilityGene TranscriptionDNA MethylationRepressive MarkCpG DinucleotidesMethyl GroupRepressive ProteinsHistone ModificationsDevelopmental ProgrammingCell DifferentiationRetroviral ElementsX-chromosome InactivationGenomic ImprintingBisulfite MutagenesisSodium BisulfiteDeaminationUracilsPCR AmplificationSequencingConversion EventsThyminesMethylated CytosinesNucleotide LevelAgarose Bead
Cite This Article
Denomme, M. M., Zhang, L., Mann, M. R. Single Oocyte Bisulfite Mutagenesis. J. Vis. Exp. (64), e4046, doi:10.3791/4046 (2012).