Name: Single Oocyte Bisulfite Mutagenesis (Video) | JoVE Journal
Uploaded: 2012-06-27T12:00:00.000+00:00
Duration: 13 min 18 s
Description: Learn single oocyte bisulfite mutagenesis for DNA methylation analysis using mouse oocytes. This method enables epigenetic studies of individual cells without pooling or contamination bias.

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5 minutes

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 °C
Lysis Solution
8 μl lysis buffer 
1 μl proteinase K 
1 μl 10% IGEPAL 
place on ice until ready for use
2:1 Agarose:Lysis Solution (10 μl per individual oocyte, amount is for 3 oocytes)
20 μl 3% LMP agarose 
10 μl Lysis Solution 
mix @ 70 °C
SDS Lysis Buffer (501 μl per individual oocyte)
<table border="0">
	<tbody>
		<tr>
			<td>1x TE pH 7.5</td>
			<td>450 μl</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>50 μl</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td> </td>
			<td>501 μl</td>
		</tr>
	</tbody>
</table>
1. Oocyte Collection
<ol>
	<li>Place the dissected mouse oviducts in M2 media, and tear the ampullae to extract the cumulus cell complex.</li>
	<li>Separate the oocytes from the cumulus cell complex using 0.3 mg/ml hyaluronidase solution in a 30 μl drop of M2 media. Keep the oocytes in solution only as long as it takes to remove the cumulus cells, as lengthy exposure may damage them. Wash the oocytes 3x in 30 μl drop of M2 media, removing cumulus cells periodically.</li>
	<li>Remove the zona pellucida using acidic tyrode's solution. Place the oocytes in one 30 μl drop of solution first, and then transfer to another 30 μl drop, as any media carried along will dilute the acid and reduce its efficiency. Keep the oocytes in solution only as long as it takes to remove the zona, as lengthy exposure may damage them. Note: an increased concentration of acidic tyrode's solution or pronase may be used for human samples, as the human zona pellucida is more resistant to treatment with acidic tyrode's solution than the mouse.</li>
	<li>Wash the oocytes once more in a 30 μl drop of M2 media.</li>
</ol>
2. Agarose Embedding and Lysis
<ol>
	<li>To perform agarose embedding, place the lysis solution on a 70 °C heatblock. Add the preheated LMP agarose to the lysis solution, producing a 2:1 agarose:lysis solution.</li>
	<li>Place a single oocyte onto a clean glass slide in minimal M2 media. Take up 10 μl of the agarose:lysis solution into a pipette tip, and (under a microscope) gently expel a small amount (~1 μl or less) onto the glass slide, allowing it to mix with the minimal media. Gently pick up the oocyte into the pipette tip and put all 10 μl into an Eppendorf tube with 300 μl mineral oil so the bead forms a sphere. 
	Note: this process must be done fairly quickly as the agarose will harden if the temperature drops as little as 5 °C below 70 °C.</li>
	<li>Incubate the tube on ice for 10 minutes. To perform lysis, remove the 300 μl mineral oil and add 500 μl of the SDS lysis buffer. Incubate overnight in a 50 °C water bath. 
	Note: Lysis solution (Table 1) may also be used for this purpose.</li>
</ol>
DAY 2
Prepare the following solutions fresh on the day of bisulfite mutagenesis. To reduce 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.
<table border="0">
	<tbody>
		<tr>
			<td>3 M NaOH</td>
			<td>2.4 g NaOH in 20 ml autoclaved ddH2O</td>
		</tr>
		<tr>
			<td>0.1 M NaOH</td>
			<td>0.5 ml of 3M in 14.5 ml autoclaved ddH2O</td>
		</tr>
		<tr>
			<td>0.3 M NaOH</td>
			<td>1.5 ml of 3M in 13.5 ml autoclaved ddH2O</td>
		</tr>
	</tbody>
</table>
2.5 M Bisulfite Solution
<ol class="lalpha">
	<li>3.8 g sodium bisulfite 
	5.5 ml GIBCO distilled H2O 
	1 ml 3 M NaOH 
	dissolve @ room temperature</li>
	<li>110 mg Hydroquinone 
	1 ml GIBCO distilled H2O 
	dissolve @ 90 °C (for only as long as it takes to dissolve, mix regularly)</li>
</ol>
When fully dissolved, mix solution (a) and (b) 
*Keep away from light*
3. Bisulfite Mutagenesis
<ol>
	<li>Fully remove the 500 μl SDS lysis buffer and add 300 μl mineral oil (~20 hours). Any lysis buffer remaining will dilute the agarose when it is heated and the bead will be more susceptible to dissolving in the subsequent steps. Proceed with bisulfite mutagenesis immediately, or store at -20 °C for up to 5 days.</li>
	<li>If applicable, remove oocytes from the freezer and let thaw (only until agarose bead is relatively translucent). Incubate for 2.5 minutes on a 90 °C heat block, following which Incubate on ice for 10 minutes. 
	Note: Do not mix or stir, extend longer than 2.5 minutes, or fluctuate temperature.</li>
	<li>To perform denaturation, remove the mineral oil and add 1 ml 0.1M NaOH to each tube, flick and invert 5-6 times.</li>
	<li>Incubate for 15 minutes in a 37 °C waterbath, inverting every 3-4 minutes. The bead should float in the NaOH.</li>
	<li>To perform bisulfite treatment, spin the tube gently, then remove the NaOH and add 300 μl mineral oil and 500 μl bisulfite solution. Incubate the tube for 3.5 hours in a 50 °C waterbath. *Keep away from light* 
	Note: Length of incubation may need to be empirically determined for gene of interest.</li>
	<li>To perform desulfonation, incubate on ice for 3 minutes, then remove the mineral oil and the bisulfite solution, spin gently, and add 1 ml 0.3 M NaOH. Flick and invert 5-6 times.</li>
	<li>Incubate for 15 minutes in a 37 °C waterbath, inverting every 3-4 minutes. The bead should float in the NaOH.</li>
	<li>Wash the samples, by first spinning gently, then remove the NaOH and add 1 ml 1x TE pH 7.5. Shake for 5-10 minutes at room temperature (on a shaker). Spin gently again, then remove the 1x TE. Repeat this washing process twice.</li>
	<li>Add 1 ml autoclaved ddH2O. Shake for 5-10 minutes at room temperature (on a shaker). Spin gently, then remove the H2O. Repeat ddH2O wash twice.</li>
	<li>Check the pH of the supernatant; it should be pH 5.0. If still too basic, wash again with H2O. Remove all supernatant, leaving only the agarose bead.</li>
</ol>
4. 1st and 2nd Round PCR amplification
<ol>
	<li>Prepare 1st round PCR mix **while washing**</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>10 μM Primer Forward Outer</td>
			<td>0.5 μl</td>
		</tr>
		<tr>
			<td>10 μM Primer Reverse Outer</td>
			<td>0.5 μl</td>
		</tr>
		<tr>
			<td>240 ng/ml tRNA</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>13 μl</td>
		</tr>
	</tbody>
</table>
Add to Illustra Ready-to-Go Hot Start PCR beads 
Carefully slide the solid agarose bead into the PCR tube (~10 μl) 
Heat to 70 °C and mix 
Add 25 μl mineral oil 
Total: 50 μl
<ol start="2">
	<li>Amplify 
	Note: An example of cycling conditions for mouse Snrpn is denaturation for 2 minutes at 94 °C, followed by 40 cycles of 30 seconds at 94 °C, 1 minute at 50 °C, and 1 minute at 68 °C; and a final 10 minute elongation step at 68 °C. Annealing temperature for 1st round PCR for mouse H19 and Peg3 is 50 °C.</li>
	<li>Prepare 2nd round PCR mix</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>10 μM Primer Forward Inner</td>
			<td>0.5 μl</td>
		</tr>
		<tr>
			<td>10 μM Primer Reverse Inner</td>
			<td>0.5 μl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>19 μl</td>
		</tr>
	</tbody>
</table>
Add to Illustra Ready-to-Go Hot Start PCR beads 
Add 5 μl 1st Round product as a template. Heat the 1st round product to 70 °C for 1 minute to soften the agarose. Be sure to pipette below the layer of mineral oil. 
Add 25 μl mineral oil 
Total: 50 μl
Note: Nested primer sequences for Snrpn, H19, and Peg3 can be found in Market-Velker et al10,12.
<ol start="4">
	<li>Amplify 
	Note: Cycling conditions for mouse Snrpn is denaturation for 2 minutes at 94 °C, followed by 40 cycles of 30 seconds at 94 °C, 1 minute at 50 °C, and 1 minute at 68 °C; and a final 10 minute elongation step at 68 °C10. Mouse H19 and Peg3 require a 50 °C annealing temperature for 2nd round PCR.</li>
	<li>As a diagnostic test, second round samples can be cut with a restriction enzyme that is methylation- or strain-specific.</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>2nd Round product</td>
			<td>4 μl</td>
		</tr>
		<tr>
			<td>Restriction Enzyme</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>4 μl</td>
		</tr>
	</tbody>
</table>
<ol start="6">
	<li>Electrophorese the digestion products on an 8% acrylamide gel. Any heterogeneous bands represent more than one sequence.</li>
</ol>
5. TA Cloning and Colony PCR
<ol>
	<li>To clone 2nd round product, first heat to 70 °C for 1 minute to soften the agarose, then ligate into vector using the Promega pGEM-T Vector System (Fisher Scientific Cat#A1360).</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>2nd Round PCR</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>pGEMT-EASY vector</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>1 μl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>2 μl</td>
		</tr>
		<tr>
			<td>2x Ligation Buffer</td>
			<td>5 μl</td>
		</tr>
	</tbody>
</table>
Incubate overnight @ 4 °C in PCR machine.
<ol start="2">
	<li>Thaw competent E.coli cells on ice for 15 minutes (Zymo Research Corp Cat#T3009). Add 3 μl ligation reaction to 8 μl E.coli and incubate ligation on ice for 15 minutes.</li>
	<li>Heat shock for 40 seconds in a 42 °C waterbath, and incubate on ice for 2 minutes. Add 60 μl S.O.C. medium and incubate at 37 °C for 1 hour (in shaker).</li>
	<li>Place all of the reaction mix on an LB/Agar/IPTG/Xgal/Amp plate and incubate plate at 37 °C overnight.</li>
	<li>Prepare colony PCR mix</li>
</ol>
<table border="0">
	<tbody>
		<tr>
			<td>20 μM M13 Forward Primer</td>
			<td>0.7 μl</td>
		</tr>
		<tr>
			<td>20 μM M13 Reverse Primer</td>
			<td>0.7 μl</td>
		</tr>
		<tr>
			<td>5X Green Go Taq Buffer</td>
			<td>7.0 μl</td>
		</tr>
		<tr>
			<td>10 mM dNTP</td>
			<td>0.7 μl</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>0.28 μl</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>25.62 μl</td>
		</tr>
		<tr>
			<td> </td>
			<td>35 μl Total</td>
		</tr>
	</tbody>
</table>
Add 35 μl Colony PCR master mix into a PCR tube. Pick a white bacterial colony from the plate with a pipette tip, and swirl it into the PCR reaction.
<ol start="6">
	<li>Amplify with denaturation for 10 minutes at 94 °C, followed by 30 cycles of 45 seconds at 94 °C, 30 seconds at 57 °C, and 1 minute at 72 °C; and a final 10 minute elongation step at 72 °C. Electrophorese 4 μl on a 1.5% agarose gel. Send ~30 μl of the PCR product for sequencing. 
	Note: For oocytes, 5 colony PCR products are sequenced.</li>
	<li>Once sequencing results are obtained, methylation patterns can be read. Any original CG that remained as a CG was methylated, and any original CG that is now a TG was unmethylated.</li>
</ol>
6. Representative Results
In our work, we assay imprinted methylation in individual oocytes and embryos (Figure 1). Following nested PCR amplification using bisulfite converted primers, it is possible to confirm a successful conversion by visualizing a correct fragment size on an agarose gel (Figure 2). An individual oocyte represents one parental allele, and in theory, has one imprinted methylation pattern. As such, second round PCR products can be tested for unintentional contamination. A restriction enzyme sensitive to DNA methylation (such as HinfI or DpnII) can be used to digest the second round PCR product to assess whether it contains a methylated or unmethylated allele (Figure 3). A methylated C within the enzyme recognition sequence is cleaved while an unmethylated C that is converted to T is no longer recognized by the enzyme and is uncut. Any MII oocyte sample containing both methylated and unmethylated alleles should be discarded, as it is indicative of cumulus cell contamination (Figure 3). Following ligation and transformation, successful colony PCR amplification can be visualized on an agarose gel to ensure samples with the correct product size are sent for sequencing (Figure 4). Finally, the sequence of five individual clones from an MII oocyte should produce five identical methylation patterns and identical nonCpG conversion rates (Figure 5a). Any samples that contain more than one pattern should be discarded (Figure 5b). Since ovulated MII oocytes have two chromosome copies or an attached polar body, there is a possibility for obtaining two similar sequence patterns (Figure 5c). We recommend discarding data from oocytes that have highly dissimilar methylation patterns since cumulus cell contamination cannot be ruled out.
 
<img alt="Oocyte isolation and bisulfite conversion process; DNA analysis diagram; bisulfite PCR." src="/files/ftp_upload/4046/4046fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Schematic of the Single Oocyte Bisulfite Mutagenesis assay.
<img alt="Gel electrophoresis result; DNA bands at 420 bp; lanes 1-4; molecular weight ladder; PCR analysis." src="/files/ftp_upload/4046/4046fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Representative results from 2nd round amplification for Snrpn from a single MII oocyte on a 1.5% agarose gel. Lanes 1-4 are four single MII oocytes and lane 5 is a negative control (no oocyte). Expected amplicon size for Snrpn is 420 bp. L, ladder.
<img alt="DNA electrophoresis gel diagram showing band separation at 420 bp, 262 bp, 103 bp, 55 bp." src="/files/ftp_upload/4046/4046fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Representative results from 2nd round methylation-specific restriction digestion for Snrpn from a single MII oocyte on an 8% acrylamide gel. HinfI diagnostic restriction digestion shows unmethylated DNA which harbors a T that abolishes the restriction site (420bp, lane 1) or methylated DNA which contains a C within recognition site (cut, 262, 103, and 54 bp, lane 2). Digestion showing both methylated and unmethylated restriction enzyme sites (cut &amp; uncut bands, lane 3) are indicative of cumulus cell contamination. L, ladder.
<img alt="DNA electrophoresis gel, 656 bp bands, showing fragment separation in lanes 1-8, ladder in lane L." src="/files/ftp_upload/4046/4046fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Representative results for colony PCR amplification for Snrpn from a single MII oocyte on a 1.5% agarose gel. Expected amplicon size following ligation of Snrpn into the pGEM-T Easy vector and using M13 forward and reverse primers is 656 bp. Lane 1-8, amplicons from clones 1-8. Clone 5 has an incorrect amplicon size and should not be sent for sequencing.
<img alt="Clonal variation analysis in DNA methylation; dot plots for methylation diversity in clones; diagram." src="/files/ftp_upload/4046/4046fig5.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5. Representative sequencing results for Snrpn from a single MII oocyte. Snrpn is methylated in oocytes. Black circles indicate methylated CpGs. White circles indicate unmethylated CpGs. CpG number and placement is representative for a B6 strain female mouse. a) Expected sequencing results for Snrpn from a single MII oocyte. Only a single strand of DNA should amplify in all five clones. Oocytes with a single methylation pattern and identical non-CpG conversion pattern should be included in analyses (percent conversion of non-CpGs indicated to the right was calculated as the number of non-CpG cytosines converted to thymine as a percentage of total non-CpG cytosines). b) Sequencing results for Snrpn from a single MII oocyte with cumulus cell contamination. Note the dissimilarity between methylation states and conversion patterns indicating multiple strand amplification. c) Sequencing results for Snrpn from a single MII oocyte with both chromosome copies or polar body inclusion.

<ol><li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+regulation+of+gene+expression%3A+how+the+genome+integrates+intrinsic+and+environmental+signals%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nat. Genet. 33, 245-254 (2003).</li>
<li>Rodenhiser, D., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+and+human+disease%3A+translating+basic+biology+into+clinical+applications%5BTitle%5D)+AND+%22CMAJ%22%5BJournal%5D)">Epigenetics and human disease: translating basic biology into clinical applications.</a> CMAJ. 174, 341-348 (2006).</li>
<li>Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 1827-1831 (1992).</li>
<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+sensitivity+mapping+of+methylated+cytosines%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((Methylation+analysis+on+individual+chromosomes%3A+improved+protocol+for+bisulphite+genomic+sequencing%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((A+bisulfite+method+of+5-methylcytosine+mapping+that+minimizes+template+degradation%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">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/pubmed?term=((DNA+Methylation%3A+Bisulphite+Modification+and+Analysis%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((A+modified+and+improved+method+for+bisulphite+based+cytosine+methylation+analysis%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((Selective+loss+of+imprinting+in+the+placenta+following+preimplantation+development+in+culture%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Dual+effects+of+superovulation%3A+loss+of+maternal+and+paternal+imprinted+methylation+in+a+dose-dependent+manner%5BTitle%5D)+AND+%22Hum.+Mol.+Genet%22%5BJournal%5D)">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/pubmed?term=((A+study+on+bisulfite+sequencing+method+for+methylation+status+of+imprinted+genes+in+single+human+oocytes%5BTitle%5D)+AND+%22Zhonghua+Yi+Xue+Yi+Chuan+Xue+Za+Zhi%22%5BJournal%5D)">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/pubmed?term=((Embryonic+imprinting+perturbations+do+not+originate+from+superovulation-induced+defects+in+DNA+methylation+acquisition%5BTitle%5D)+AND+%22Fertil.+Steril%22%5BJournal%5D)">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/pubmed?term=((Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li>
<li>Hajj, N. E. l <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limiting+dilution+bisulfite+%28pyro%29sequencing+reveals+parent-specific+methylation+patterns+in+single+early+mouse+embryos+and+bovine+oocytes%5BTitle%5D)+AND+%22Epigenetics%22%5BJournal%5D)">Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes.</a> Epigenetics. 6, 1176-1188 (2011).</li>
</ol>

The authors have nothing to disclose.

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

single oocyte bisulfite mutagenesis

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.

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 ...

The overall goal of this procedure is to analyze DNA methylation in a single oocyte. This is accomplished by first removing cumulus cells and soap palita from collected oocytes. The second step is to embed the oocyte in a two to one aros lysis solution to protect the minute amount of DNA from being lost or degraded.Next bi sulfite mutagenesis is performed, followed by amplification through nested polymerase chain reaction. The final step is to ligate and clone the PCR product. Clones are then picked PCR amplified and sent for sequencing.Ultimately, bi sulfite mutagenesis and sequencing is used to show DNA methylation at cytosine phosphate guine, or CPG dinucleotides of a region of interest in a single oocyte. The main advantage of this technique over existing methods like bi sulfite mutagenesis of large cell numbers, is that data can be obtained from a single oocyte without pooling cytes PCR bias and chemo cell contamination. On the day of oocyte collection, freshly prepare the solutions listed in the written protocol when making solutions reduce the chance of DNA contamination by changing gloves often and using filter tips also keep tubes angled away when open and recap all tubes when not in use.Begin OSI collection by placing the dissected mouse ovaducts into M two media and ripping the M pulley to extract the cumulus cell complex. Then separate the cytes from the cumulus cell complex using 0.3 milligrams per milliliter of hyaluronidase solution in a 30 microliter drop of M two media. Keep the cytes in solution only as long as it takes to remove the cumulus cells as lengthy exposure may damage them.Wash the oocytes in 30 microliter drops of M two media by transferring the oocytes from drop to drop. Removing the cumulus cells periodically for a total of three washes. Remove the Z of LUCITA using acidic tyro solution.First place the oocytes in one 30 microliter drop of solution, then transfer to another 30 microliter drop. As any media carried along will dilute the acid and reduce its efficiency. Keep the cytes in solution only as long as it takes to remove the zona as lengthy exposure may damage them.Following removal of the zona, wash the cytes again in a 30 microliter drop of M two media to perform agros embedding first place the lysis solution on a 70 degree Celsius heat block. Add the preheated low melting point, or LMP Agros to the lysis solution, producing a two to one AGROS to lysis solution. Then place a single oocyte onto a clean glass slide in minimal M two media.The next step is to embed the single cyte into the Agros and lysis solution. This must be done quickly as the agros will harden at temperatures below 65 degrees Celsius. Take up 10 microliters of the Agros lysis solution into a pipette tip under a microscope.Gently expel about one microliter or less of the agros lysis solution onto the glass slide, allowing it to mix with the minimal media. Gently pick up the oocyte into the pipette tip. Then put all 10 microliters into an einor tube with 300 microliters of mineral oil so that the resulting bead takes on a spherical shape.Incubate the tube on ice for 10 minutes to perform lysis. Remove the 300 microliters of mineral oil and add 500 microliters of the SDS lysis buffer. Incubate the sample overnight in a water bath set to 50 degrees Celsius on the second day.Prepare the bi sulfide mutagenesis solutions fresh as described in the written protocol. Begin mutagenesis by fully removing the 500 microliters of SDS lysis buffer. Keep in mind that any lysis buffer remaining will dilute the agros when it is heated and the bead will be more susceptible to dissolving in the subsequent steps.Following complete removal of the lysis buffer, add 300 microliters of mineral oil. Incubate the sample for two and a half minutes on a 90 degree Celsius heat block. Take carat to mix or stir, extend, or fluctuate the temperature.After two and a half minutes. Incubate the sample on ice for 10 minutes. To perform denaturation, remove the mineral oil and add one milliliter of 0.1 molar sodium hydroxide to the tube flick and invert the tube five to six times.Incubate the sample for 15 minutes. In a water bath set to 37 degrees Celsius inverting every three to four minutes, the bead should float in the sodium hydroxide. Note that the bead is no longer at the bottom of the tube.To perform by sulfite treatment first spin the tube gently following removal of the sodium hydroxide. Add 300 microliters of mineral oil and 500 microliters of BIS sulfite solution. Take care to avoid exposure to light and incubate the tube for three and a half hours in a 50 degrees Celsius water bath.Now perform desination. First incubate the sample on ice for three minutes before removing the mineral oil and BIS sulfite solution. After a gentle spin, add one milliliter of 0.3 molar sodium hydroxide flick and invert the tube five to six times.Incubate the sample for 15 minutes. In a 37 degree Celsius water bath inverting every three to four minutes, again, the bead should float in the sodium hydroxide prior to beginning PCR. Wash the samples by first spinning gently.Then remove the sodium hydroxide and add one milliliter of one xte pH 7.5. Shake for five to 10 minutes at room temperature. Following a second gentle spin, remove the one XTE.Repeat this washing process twice. Then add one milliliter of autoclave, double distilled water. Shake for five to 10 minutes at room temperature, spin gently for a third time and remove the water.Repeat the double distilled water wash twice. Finally, remove all supernatant, leaving only the aros bead. Check that the pH of the supernatant is 5.0.If the supernatant is still too basic, wash the beads again with double distilled water. Meanwhile, add the first round PCR mix to the PCR tube as described in the written protocol. Slide the solid agro speed to the lip of the einor tube.Then carefully transfer the agro speed to the PCR tube heat to 70 degrees Celsius for one minute and mix then amplify is suggested in the written protocol. Following first round amplification, prepare the second round PCR reaction using five microliters of the first round product as a template. Be sure to bipa below the layer of mineral oil.Proceed to perform the second round of amplification as directed in the text as a diagnostic test. Second round samples can be cut with a restriction enzyme that is methylation or strain specific electrophoresis the digestion products on an 8%acrylamide gel. Any heterogeneous bands that result represent more than one sequence to clone the second round product ligate into the vector and perform TA cloning as described in the manuscript accompanying this video following TA cloning plate, all of the reaction mix onto an agar and LB plate, supplemented with ampicillin, ex gal and IPTG and incubate the plate at 37 degrees Celsius overnight.Then add the colony PCR master mix to the PCR tube as listed in the written protocol. Pick a white bacterial colony from the plate with a pipette tip and swirl it into 35 microliters of the colony PCR master mix in a PCR tube. Proceed to amplify the suspension as described in the text following PCR amplification electrophoresis four microliters of the PCR product on a 1.5%AGROS gel.Finally, send approximately 30 microliters of the PCR product for sequencing of five colony PCRs per cyte. Once sequencing results are obtained, methylation patterns can be read any original cytosine guanine that remained as such was methylated. Conversely, any original cytosine guanine that is now a thiamine guanine was unmethylated following nested PCR amplification using bi sulfite converted primers.It is possible to confirm a successful conversion by visualizing a correct fragment size on an aros gel individual metaphase. Two oocyte represents one parental allele and in theory has one imprinted methylation pattern As such. Second round PCR products can be tested for unintentional contamination.A restriction enzyme sensitive to DNA methylation can be used to digest the second round PCR product to assess whether it contains a methylated or unmethylated allele. Here, a methylated cytosine within the enzyme recognition sequence is cleaved. While an unmethylated cytosine that is converted to thymine is no longer recognized by the enzyme and is uncut any metaphase two oocyte sample containing both methylated and unmethylated alleles should be discarded as it is indicative of cumulus cell contamination or polar body inclusion Following ligation and transformation.Successful colony PCR amplification can be visualized on an AROS gel to ensure samples with the correct product size are sent for sequencing. For this example, the expected amplicon size following ligation is hundred 56 base pairs. Here, clone five has an incorrect amplicon size and should not be sent for sequencing.The sequence of five individual clones from a metaphase two oocyte should produce five identical methylation patterns and identical non CPG conversion rates. Any samples that contain more than one pattern should be discarded. Since ovulated metaphase two cytes have two chromosome copies or an attached polar body, there is a possibility for obtaining two similar sequence patterns.Data should be discarded from any cytes that have highly dissimilar methylation patterns. Since cumulus cell contamination cannot be ruled out When attempting this procedure, it is important to remember first to wash oocytes multiple times in fresh medium, following HI IDE treatment to remove as many cumul cells as possible. Secondly, the low melting point agros will harden at temperatures below 65 degrees Celsius, so work carefully but quickly.And finally, sodium bis sulfate and hydroquinone are light sensitive, so reaction tubes and solutions should be wrapped in foil.

Research

JoVE Journal

Biology

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation.  Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome.  Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Figure 1: End-to-end framework. The figure illustrates end-to-end framework for multimodal knowledge Graph VISHAM-KG. Please click here to view a larger version of this figure.

Preparation of Meiotic Chromosome Spreads from Mouse Oocytes for Assessment of Synapsis and Recombination

Learn single oocyte bisulfite mutagenesis for DNA methylation analysis using mouse oocytes. This method enables epigenetic studies of individual cells without pooling or contamination bias.

Single Oocyte Bisulfite Mutagenesis

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