Rapid sexing of murine preimplantation embryos using a nested, multiplex polymerase chain reaction (PCR).

摘要

The development of a rapid polymerase chain reaction (PCR) procedure for sexing mouse preimplantation embryos at the 4- to 8-cell stage is described. Primers to amplify the X-chromosome microsatellite locus (DXNds3) and the Y-chromosome Sry and Zfygenes were used to develop the nested, multiplex PCR assay. The sensitivity of the assay was measured using groups of 4, 2 and 1 blastomere(s) from dissociated embryos. The efficiency of the assay was evaluated in single blastomeres obtained by embryo biopsy. The accuracy of sexing was determined by comparing single-cell results with those from matched blastocysts. Robust amplification of male (XY) and female (XX) gene sequences was obtained in less than 6 h. The percentage of male (3 bands) and female(1 band) reactions for groups of 4, 2 and 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 10 male and 13 female embryos, sexing of single blastomeres accurately (100%) predicted results of matched blastocysts. Simultaneous amplification of one X- and 2 Y-gene sequences ensured correct interpretation of sexing reactions. Short terminal cycling times and minimal tube handling increased the assay speed and decreased the potential risk of contamination.