Одноместный мутагенеза бисульфит ооцитов
Summary
Бисульфит мутагенеза является золотым стандартом для анализа метилирования ДНК. Наш модифицированный протокол позволяет для анализа ДНК метилирования на уровне одной клетки и была специально разработана для отдельных яйцеклеток. Она также может быть использован для расщепления стадии эмбриона.
Abstract
Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.
Protocol
Discussion
Это один анализ ооцитов содержит много шагов, число которые имеют решающее значение и требуют особого ухода. Первый яйцеклетки стирки. Это особенно важно мыть каждую яйцеклетку несколько раз в свежей среде падает после гиалуронидазы лечение, чтобы удалить как многие клетки кучевые на?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Эта работа была поддержана в Университете Западного Онтарио, кафедра акушерства и гинекологии, а также грант ER06-02-188 от исследований и инноваций Ministryof, ранний премии научный сотрудник. MMD была поддержана Программой CIHR Обучение в репродукции, раннему развитию и воздействие на здоровье (REDIH) Высшее стипендии.
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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