| Abstract: | 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. Mol. Reprod. Dev. 49:261–267, 1998. © 1998 Wiley-Liss, Inc. |
