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The objective of this study was to develop a rapid and efficient means of sexing murine preimplantation embryos at the 4- to 8-cell stage of development. To achieve this goal, a nested, multiplex polymerase chain reaction (PCR) was optimized using DNA from male and female mice and primers specific for X- (DXNds3)- and Y- (Sry,Zfy) gene sequences. Sensitivity of the assay was measured using groups of 4, 2, or 1 blastomere from dissociated embryos. Efficiency was evaluated using single blastomeres obtained by embryo biopsy. Accuracy of sexing was determined by comparing single-cell results with those of matched blastocysts. Robust amplification of male (XY) and female (XX) gene sequences was obtained in less than 6 hours. The percentage of male (3 bands) and female (1 band) reactions for groups of 4, 2, or 1 blastomere was 100% (6/6), 100% (15/15), and 94.4% (17/18), respectively. Assay efficiency for single, biopsied blastomeres from 4 to 8 cell embryos was 95.8% (207/216). For male and female embryos, sexing of single blastomeres accurately predicted results of matched blastocysts, 100% (10/10) and 100% (13/13), respectively. Simultaneous amplification of one X- and two Y-gene sequences ensured correct interpretation of sexing reactions. Short thermal cycling times and minimal tube handling increased the assay speed and decreased the potential risk of contamination. |
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