Single Oocyte Bisulfite Mutagenesis

Summary

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.

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 reported^4-6 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

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 ....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog 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
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