This work was supported by the University of Western Ontario, the Department of Obstetrics and Gynaecology; and a grant ER06-02-188 from the Ministryof Research and Innovation, Early Researcher Award. MMD was supported by a CIHR Training Program in Reproduction, Early Development and the Impact on Health (REDIH) Graduate Scholarship.

DAY 1
Prepare the following solutions fresh on the day of oocyte collection with sterile, distilled water such as GIBCO water. To reduce the chance of DNA contamination, change gloves often and use filter tips. Keep tubes angled away when open, and recap all tubes when not in use. We recommend that solutions are made as n+1.
3% LMP Agarose
30 mg Low Melting Point (LMP) Agarose 
up to 1 ml GIBCO H2O 
dissolve @ 70 &deg;C...

<ol>
	<li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nat. 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L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Feil, R., Charlton, J., Bird, A. P., Walter, J., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+analysis+on+individual+chromosomes:+improved+protocol+for+bisulphite+genomic+sequencing.">Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing.</a> Nucleic Acids Res. 22, 695-696 (1994).</li><li>Raizis, A. M., Schmitt, F., Jost, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bisulfite+method+of+5-methylcytosine+mapping+that+minimizes+template+degradation.">A bisulfite method of 5-methylcytosine mapping that minimizes template degradation.</a> Anal. Biochem. 226, 161-166 (1995).</li><li>Patterson, K., Molloy, L., Qu, W., Clark, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Methylation:+Bisulphite+Modification+and+Analysis.">DNA Methylation: Bisulphite Modification and Analysis.</a> J. Vis. Exp. (56), e3170 (2011).</li><li>Olek, A., Oswald, J., Walter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+and+improved+method+for+bisulphite+based+cytosine+methylation+analysis.">A modified and improved method for bisulphite based cytosine methylation analysis.</a> Nucleic Acids Res. 24, 5064-5066 (1996).</li><li>Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+imprinting+in+the+placenta+following+preimplantation+development+in+culture.">Selective loss of imprinting in the placenta following preimplantation development in culture.</a> Development. 131, 3727-3735 (2004).</li><li>Market-Velker, B. A., Zhang, L., Magri, L. S., Bonvissuto, A. C., Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+effects+of+superovulation:+loss+of+maternal+and+paternal+imprinted+methylation+in+a+dose-dependent+manner.">Dual effects of superovulation: loss of maternal and paternal imprinted methylation in a dose-dependent manner.</a> Hum. Mol. Genet. 19, 36-51 (2010).</li><li>Meng, L. H., Xiao, S. Q., Huang, X. F., Zhou, Y., Xu, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+on+bisulfite+sequencing+method+for+methylation+status+of+imprinted+genes+in+single+human+oocytes.">A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes.</a> Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 25, 289-292 (2008).</li><li>Denomme, M. M., Zhang, L., Mann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+imprinting+perturbations+do+not+originate+from+superovulation-induced+defects+in+DNA+methylation+acquisition.">Embryonic imprinting perturbations do not originate from superovulation-induced defects in DNA methylation acquisition.</a> Fertil. Steril. 96, 734-738 (2011).</li><li>Tahiliani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li><li>Hajj, N. 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This single oocyte assay contains many steps with a number that are critical and require special care. The first is oocyte washing. It is particularly important to wash each oocyte multiple times in fresh medium drops following hyaluronidase treatment to remove as many cumulus cells as possible. Moreover, when transferring oocytes to acidic tyrode's solution for zona pellucida removal make sure surrounding medium is clear of cumulus cells. The oocyte is very sticky following zona removal, and any surrounding cumulus cell...

Table of specific reagents and equipment.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Oocyte Collection</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acidic Tyrode</td>
			<td>Sigma</td>
			<td>T1788</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma</td>
			<td>P5568</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10% IGEPAL</td>
			<td>Bioshop</td>
			<td>NON999.500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">Lysis Solution</td>
		</tr>
		<tr>
			<td>Tris pH 7.5</td>
			<td>Bioshop</td>
			<td>TRS001.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sigma</td>
			<td>L9650</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA pH 8.0</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LiDS</td>
			<td>Bioshop</td>
			<td>LDS701.10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Invitrogen</td>
			<td>P2325</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">SDS Lysis Buffer</td>
		</tr>
		<tr>
			<td>TE pH 7.5</td>
			<td>
			Bioshop(Tris)
			Sigma (EDTA)</td>
			<td>
			TRS001.5
			E5134</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Bioshop</td>
			<td>SDS001.500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">Bisulfite Conversion</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Hydrogensulfite (Sodium Bisulfite)</td>
			<td>Sigma</td>
			<td>243973</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma</td>
			<td>H9003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Low Melting Point (LMP) Agarose</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M2 Medium</td>
			<td>Sigma</td>
			<td>M7167</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GIBCO Distilled water</td>
			<td>Invitrogen</td>
			<td>15230-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoclaved double distilled (dd) water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">PCR</td>
		</tr>
		<tr>
			<td>Illustra Hot Start Mix RTG</td>
			<td>GE Healthcare</td>
			<td>28-9006-54</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>240 ng/ml yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-011</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5x Green GoTaq Reaction Buffer</td>
			<td>Promega</td>
			<td>M7911</td>
			<td>&nbsp;</td>
		</tr>
<tr>
			<td>Inner and outer nested primers</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">Ligation</td>
		</tr>
		<tr>
			<td>Promega pGEM-T Easy Vector</td>
			<td>Fisher Scientific</td>
			<td>A1360</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">TA Cloning</td>
		</tr>
		<tr>
			<td>Competent E.coli cells</td>
			<td>Zymo Research Corp.</td>
			<td>T3009</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70&deg;C and 90&deg;C Heat Blocks</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C and 50&deg;C Waterbaths (42&deg;C for transformations)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

single oocyte bisulfite mutagenesis

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.

Watch this Scientific Journal Video about Single Oocyte Bisulfite Mutagenesis at JoVE.com

Single Oocyte Bisulfite Mutagenesis

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Research

JoVE Journal

Biology

13.9K ビュー.  Schulich School of Medicine and Dentistry, University of Western Ontario. 亜硫酸塩の突然変異誘発は、DNAメチル化を解析するためのゴールドスタンダードです。私たちの変更されたプロトコルは、単一細胞レベルでのDNAメチル化解析を可能にし、特に個々の卵のために設計されました。また、開裂段階の胚に使用することができます。