Reliable sex determination of mouse preimplantation embryos by PCR amplification of male-specific genes in single blastomeres.

OBJECTIVE: To assess the reliability of sex determination in mouse preimplantation embryos using the two-step polymerase chain reaction method. SETTING: Division of Immunology, Department of Microbiology and Division of Reproductive Medicine, Department of OB/GYN. METHODS: The Sry and Zfy genes, known to be present in the sex-determining region of mouse Y chromosome, were selected for Y-specific target sequences and DXNds 3 locus located on mouse X chromosome was served as the internal control sequence. DNAs extracted from heart blood of male and female mice were used to test the correctness and specificity of the selected primers using the two-step PCR method. The same experimental conditions were then used to amplify the single copy genes in single mouse blastomeres with two pairs of primers for each of the target sequences. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. RESULTS: All male and female blood DNA sample results confirmed the correct sex identification of the origin (100%). Nineteen of 20 single blastomeres showed the accurate diagnosis when compared with theirs 7/8 embryos. The sex of 36 of 37 mouse pups born from biopsied male and female embryos agreed with the predicted sex. CONCLUSION: The reliable genetic analysis of sex chromosome- specific sequences in single cell is possible by the two-step PCR method and could be applied for diagnosis of defective genes of human preimplantation embryos derived from the in vitro fertilization program.