Sexing and multiple genotype analysis from a single cell of bovine embryo

We described a procedure for multiple genotype analysis (determination of s ex and of three genetic markers) from a single cell derived from bovine pre implantation embryo. It consists of primer extension preamplification-polym erase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR . A single blastomere that was isolated by microaspiration from bovine embr yos at the 16- to 32-cell stage then was lysed and was subjected to the PEP -PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa -casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91., 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa -casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0. 05), as was seen by obtaining a similar percentage of embryos developing fu rther into the blastocyst stage in the group subjected to biopsy (44/75, 59 %) and in the control group of embryos (30/50, 60%). (C) 2001 by Elsevier S cience Inc.