102 ACCURACY OF PRE-IMPLANTATION GENETIC DIAGNOSIS USING CELLS BIOPSIED FROM EQUINE BLASTOCYSTS

Abstract

There is growing interest in pre-implantation genetic diagnosis (PGD) for management of inherited genetic disease in the horse. In a previous study (Choi et al. 2010 Reproduction 140, 893–902), we demonstrated normal viability of equine blastocysts after biopsy. However, genome amplification was only moderately successful and only 1 of 2 analyses of heterozygous loci accurately detected both alleles. In the current study, we investigated different methods for amplification of DNA to improve the efficiency of PGD. To evaluate allele drop-out, multiple commonly-heterozygous gene loci were evaluated. In Experiment 1, using a piezo drill, 3 to 5 biopsy samples of 20 to 30 cells each were obtained from each of 4 in vitro-produced blastocysts. The samples and embryos were stored at –20°C, then shipped to the Veterinary Genetics Laboratory at the University of California, Davis. Whole genome amplification was done with an Illustra Genomiphi V2 kit (GE Healthcare, Waukesha, WI) before PCR for specific markers. Two disease-related (SCN4A and PPIB), one gender (AME) and 17 microsatellite identification markers were genotyped, for a total of 20 loci. Results for biopsy samples were compared with those for the corresponding embryo. A DNA signal was obtained from 14/15 biopsy samples, but for only 59.6% of the 280 total genotypes. Of 40 heterozygous loci, the signal from the corresponding biopsy sample showed only one allele (underwent allele dropout) in 60/80 instances (75%). In Experiment 2, 4 biopsies were obtained from each of 4 additional in vitro-produced blastocysts, then all samples were stored at –20°C. The Repli-G Mid kit (Qiagen, Valencia, CA) was used for whole genome amplification. Two disease-related (SCN4A and PPIB), 2 gender (AME and eSRY), 10 coat colour and 17 identification markers (total of 31 loci) were examined in each biopsy sample and were compared with results for the embryos. One biopsy sample was lost. Signal was obtained from 14/15 of the remaining biopsy samples and gave a 100% match at the 2 gender loci, 2 disease-related loci and 10 coat colour loci. One identification locus, LEX33, amplified in only 8 of 22 analyses. At the remaining 16 identification loci, 223/224 biopsy results matched those for the embryos. Overall, of 51 heterozygous loci among the 4 embryos, biopsy samples exhibited allele dropout in 1/180 instances (0.6%). In conclusion, results obtained using piezo-driven embryo biopsy and whole genome amplification using the Qiagen Repli-G kit have high accuracy and this technique may be suitable for use in a clinical setting. Further studies are needed with in vivo-derived embryos and to optimize accuracy of PCR of some identification markers.

This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University and by Ms Kit Knotts.