330 UTILIZING PRE-IMPLANTATION GENETIC DIAGNOSIS AND OPU-IVP-ET TO GENERATE MULTIPLE PROGENY OF PREDETERMINED GENOTYPE FROM CLONED TRANSGENIC HEIFERS

Abstract

Advanced reproductive technologies, comprising ovum pickup (OPU), in vitro embryo production (IVP), and embryo transfer (ET), were used to rapidly generate multiple F1 offspring from nine hemizygous founder-cloned transgenic (TG) heifers that highly express casein protein in their milk (Brophy B et al. 2003 Nat. Biotech. 21, 157–62). Moreover, embryo biopsy and genotyping were used to determine gender and presence or absence of the additional casein genes prior to ET. This minimized the number of surplus animals that would otherwise be generated from the breeding program. The aim was to generate 10 TG female (TG-F), 10 non-TG female (non-TG-F), two TG male (TG-M), and two non-TG male (non-TG-M) offspring. Some hemizygous TG bulls were required to enable the generation of homozygous transgenic animals in the next generation, while non-TG cattle would serve as controls in future studies. Transvaginal OPU was performed on non-stimulated TG heifers twice weekly for 6 weeks. Selected oocytes were matured, then fertilized in vitro (Day 0) with sperm from a conventional sire, and cultured to Day 6 (Thompson JG et al. 2000 J. Reprod. Fert. 118, 47–55). Transferable quality embryos were biopsied and cultured singularly for another 20 h, by which time genotyping results were available. On Day 7, individual embryos of the desired genotype were identified and transferred nonsurgically to synchronized recipients. Two biopsy methods were used, dependent on the stage of the embryo. For blastocysts, 10–12 trophectoderm cells were biopsied using an ultra-sharp splitting blade mounted on a micromanipulator (Herr CM et al. 1991 Theriogenology 35, 45–54). For morulae, the zonae pellucidae were enzymatically removed and two blastomeres per embryo aspirated with a blunt 30-μm micropipette (Chrenek P et al. 2001 Theriogenology 55, 1071–81). Aliquots either from the lysed blastocyst biopsy samples or following primer extension pre-amplification of DNA (Zhang L et al. 1992 Proc. Natl. Acad. Sci. USA 89, 5847–51) from blastomere samples were used to determine sex and casein transgene status using multiplex PCR. Following the OPU program, the TG heifers were artificially inseminated with semen from the same sire as used for IVF; eight calved (89%). Data were compared using Fisher's exact test. The mean number of transferable Day 6 embryos produced per donor per OPU session was 1.3. Pregnancy rates following OPU-IVP-ET on Day 35 were 27/62 (44%) with a calving rate of 29%. Nine TG-F, five non-TG-F, three TG-M, and one non-TG-M were delivered vaginally at term. All calves born were of the predicted genotype. There was no significant difference in embryonic survival to term either between TG and non-TG embryos transferred, or between biopsy methods. One OPU calf died shortly after birth; the other 17 survived to weaning, as did all the AI calves. The results show that pre-implantation genetic diagnosis combined with OPU-IVP-ET can be successfully used to produce small scale-up herds from founder TG females.