The goal of this project is to develop a system for rapid determination of the genetic sex of mouse embryos. The strategy will be to biopsy the 6-8 - cell conceptus and determine the sex of the biopsied blastomere as the remainder of the embryo cleaves to the blastocyst stage. This rapid diagnostic procedure will thus allow determination of sex before it is necessary either to freeze or implant the embryo. Such a capability would have significant impact in agriculture, where sexing is economically important, and may also be eventually applied to diagnosis of muscular dystrophy. The advantage of such a rapid diagnosis is that embryo mortality associated with freezing can be avoided. The technique for mouse embryo sexing will be based upon the polymerase chain reaction (PCR). Single blastomeres will be removed from embryos, boiled, and annealed with synthetic oligonucleotides corresponding to a Y-chromosome specific repeated DNA element. The oligonucleotides will serve as primers for copying a short intervening portion of the Y repeat. After repeated rounds of annealing, polymerization in the presence of 32P-dCTP, and denaturation, the PCR reaction mixture will be subjected to acrylamide gel electrophoresis. The acrylamide gel will be then be dried and exposed directly to X-ray film, thus eliminating the time-consuming procedure of Southern hybridization. Those blastomeres which exhibit amplification of the Y-chromosome specific element will be diagnosed as males, and negative embryos will be scored as females. To confirm success of the procedure, the corresponding blastocysts will be implanted into the uteri of pseudopregnant females. Fetuses which develop will be examined to determine if their phenotypic sex corresponds to that predicted by PCR. Development of this technique will not only allow rapid determination of embryo sex, it will establish a protocol for evaluation of any genetic locus, regardless of whether that locus is expressed during preimplantation development. Thus, continued refinement of the process could eventually permit a complete genetic analysis of embryos, with the consequence that genetic diseases of any kind will be diagnosable prior to implantation.
