Mutagenesis, Vol. 14, No. 4, 437-438,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Short Communication |
Modification of the Comet assay for the detection of DNA strand breaks in extremely small tissue samples
Department of Dermatology, University of California, 1 Reproductive Genetics Unit, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA 94143, USA and 2 Novartis Pharma AG, Genetic Toxicology, WSH 2881.5.14, CH-4002 Basel, Switzerland
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Abstract |
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We modified the Comet assay to enable the quantification of DNA strand breakage in individual cells of extremely small tissue samples. This modification was used to analyze cells isolated from the ectoplacental cone and egg cylinder of mouse embryos at embryonic day 7.5. We detected more naturally occurring DNA strand breaks and a higher number of apoptotic cells in the ectoplacental cone compared with the egg cylinder.
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Introduction |
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The Comet assay (single cell gel electrophoresis) is a sensitive technique for the detection of single- and double-strand breaks and alkaline-labile sites in nuclear DNA of individual cells. The Comet assay has the added advantage of detecting apoptotic/necrotic cells, which can be clearly distinguished from viable cells exhibiting DNA strand breaks (Olive et al., 1998
). This assay has been used to measure DNA strand breaks in embryo-derived cell cultures, such as mouse embryonic fibroblasts (Connor et al., 1997) and mouse embryonic stem cells (Vatolin et al., 1997), as well as adult animal tissue samples (Latinwo et al., 1997). However, it has not yet been adapted to very small sample volumes such as those found in early stage mouse embryos. We describe a modification of the Comet assay to measure DNA strand breaks in embryonic tissues.
For the Comet assay we used the standard procedure originally described by Singh et al. (1988) with modifications (Klaude et al., 1996; Hartmann et al., 1998). We found that the standard procedure, which entailed mincing tissues, resuspending in phosphate-buffered saline (PBS) and pelleting cells prior to resuspension in agarose resulted in a very low recovery rate of cells from early stage embryos. Therefore, we modified the procedure so as to mince the tissue directly on microscope slides. In this procedure, mouse embryos [C57BL/6J x (tac:N:NIHS-BC x 129 SvJ)] were recovered, washed in cold PBS, separated into egg cylinders and ectoplacental cones (giant trophoblasts and parietal endoderm were removed and discarded) and then aspirated with 5 µl cold PBS and pipetted onto slides. The slides had been coated with a thin layer of agarose and allowed to dry. The tissue was minced into small pieces using two 27 gauge needles. This procedure did not damage the coating of the slide significantly. The pieces were then suspended in 40 µl of 0.5% low melting point agarose and pipetted back onto the slide. This procedure yielded a good amount of individual cells. The agarose suspension was covered with a 25x25 mm coverslip and placed at 4°C for 5 min. The coverslip was gently removed and the slide was submersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10) for at least 1 h. After lysis, the slides were equilibrated for 20 min in a jar containing alkaline buffer (300 mM NaOH, 1 mM EDTA, pH >13), transferred into an electrophoresis unit with alkaline buffer and subjected to an electric field of 0.86 V/cm for 10 min. Following electrophoresis the microgels were neutralized in 0.4 M Tris (pH 7.5), rinsed with water, dehydrated in 100% ethanol for 2 min and allowed to dry at room temperature. The DNA was stained with YOYO®-1 (Molecular Probes, Eugene, OR) (1:1000 in antifade) and visually examined on a fluorescence microscope.
To avoid slide-to-slide variation of results, the ectoplacental cone and the egg cylinder from the same embryo were placed on the same slide. We found that prolonged periods under the microscope to dissect embryos into individual tissue layers (endoderm, mesoderm and ectoderm) resulted in unreliable data (results not shown). Thus, it is important that the tissues be kept cold and the samples be processed expeditiously. Furthermore, treating embryos with pancreatin/trypsin solution, a mild lysis buffer used to assist in microdissection, resulted in high background levels of DNA breakage (presumably due to DNases in pancreatin). Analysis of DNA strand breakage was performed according to Collins et al. (1993). Based on the extent of strand breakage, each cell was assigned to one of five classes, ranging from 0 (no visible tail) to 4 (still a detectable head of the comet but most of the DNA in the tail). Additionally, cells with a very small head region or without a detectable head were classified as apoptotic according to Olive et al. (1998). However, the Comet assay is not suited to accurately quantify apoptotic cells because highly fragmented DNA in late apoptotic cells is expected to migrate out of the gel. We therefore decided to calculate percentages of cells showing the DNA breakage classes 0–4 and to score cells that show the morphology of apoptotic cells as described by Olive et al. (1998) separately.
Figure 1 compares DNA strand breaks detected by the Comet assay in cells derived from mouse egg cylinders and ectoplacental cones. A total of 13 embryos from two litters (2768 individual cells) were analyzed. Cells from the egg cylinders showed a bell-shaped distribution with most cells assigned to comet classes 2 and 3. In contrast, ectoplacental cones showed most cells in the highest comet classes (classes 3 and 4), indicating extensive DNA breakage. Furthermore, ectoplacental cones contained many more apoptotic cells than did egg cylinders (30 and 5%, respectively, of the total cell count).
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Most DNA repair assays require cell cultures and are unamenable to small sample sizes. Because the Comet assay measures DNA breaks in individual cells it can be adapted for use on very small samples. In this report we have demonstrated the use of the Comet assay on embryonic day 7.5 (E7.5) mouse egg cylinders, which contain ~5000–15 000 cells (Power and Tam, 1993
), and on ectoplacental cones, which contain even fewer cells. We have also successfully applied the Comet assay to earlier stage embryos (E6.5) and to tissue layers (endoderm, mesoderm and ectoderm) microdissected from E7.5 embryos (data not shown). This protocol can, therefore, be used to determine the dynamics of DNA breaks during embryonic development. Furthermore, several DNA repair knockout mice have recently been constructed that die in early developmental stages, i.e. RAD51–/–, Brca1–/–, APE–/– and Xrcc1–/– embryos (for a review of DNA repair knockout mice see Tebbs et al., 1998). The Comet assay could be used to measure DNA damage in individual repair-deficient mouse embryos prior to embryonic death. In our studies, we detected a relatively high amount of DNA strand breaks in cells from early mouse embryos, which may, in part, be due to ongoing differentiation processes. In control experiments using liver and gastric mucosa tissues prepared in an identical fashion (i.e. tiny amounts of tissue were minced on the slides), the majority of cells were in classes 0 and l (results not shown), showing that this method does not introduce additional DNA damage. Therefore, the relatively high amounts of strand breaks in embryonic tissue, as also demonstrated by Vatolin et al. (1997) in embryonic stem cells following in vitro differentiation, might be a characteristic of embryonic cells. This issue will be further discussed elsewhere (Tebbs et al., 1999).
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Acknowledgments |
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This work was supported in part by NIH grant ES08750.
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Notes |
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3 To whom correspondence should be addressed. Tel: +41 61 324 1951; Fax: +41 61 324 1274: Email: andreas.hartmann{at}pharma.novartis.com
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Received on December 7, 1998; accepted on March 10, 1999.
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