PRODUCTION OF LIVE OFFSPRING WITH PREDICTED GENOTYPES USING PCR-RFLP ANALYSIS OF POLAR BODIES FROM MOUSE OOCYTES

Accurate identification of genotypes in gametes and early embryos coul d facilitate the efficient production of offspring with desirable trai ts, This study demonstrates the feasibility of producing offspring wit h predictable genotypes from micromanipulated mouse oocytes. The Polym erase Chain Reaction (PCR) was used to amplify genes in the IA subregi on of the major histocompatibility complex of the mouse. The validity of the approach was demonstrated in experiment 1 with IA haplotypes of unfertilized mouse ova amplified via PCR and distinguished by restric tion fragment length polymorphism (RFLP) analysis. In experiment 2, fe rtilized oocytes were micromanipulated to remove the first and second polar bodies, which were then genotyped by validated PCR-RFLP procedur es. Primary oocytes of heterozygous females contain two copies of each of the different alleles. Following meiosis I and II, the genotype of the ovum was predicted by subtracting the alleles observed in microma nipulated polar body samples. Sixty-two fertilized ova were micromanip ulated and transferred to recipient females resulting in 27 live offsp ring (44%). The correct maternal contribution to the embryonic genotyp e was predicted in 19 of 27 (71%) offspring as confirmed by PCR-RFLP a nalysis of DNA from pup tails. Predicted genotypes of two pups were no t confirmed (7%), whereas no prediction could be made in six cases (22 %). (C) 1995 Wiley-Liss, Inc.