Figure 1.

Genetic engineering of pigs by embryo microinjection. Estrus synchronization of potential embryo recipient (surrogate pigs) and donor pigs is achieved by a refined hormone regimen. A variation in the regimen is utilized in the embryo donors to increase ovulation rate and the potential for higher yield of one-cell embryos (zygotes). The embryo donors are artificially inseminated on second estrus detection and euthanized shortly thereafter for collection of single-cell presumptive zygotes. The isolated zygotes are microinjected with the CRISPR/Cas9 components and implanted into the oviducts of surrogate pigs by surgical access usually within 4–6h of initial embryo collection. Transabdominal ultrasound is used to confirm pregnancy and to track fetal development. Shortly after parturition, genomic DNA is obtained from the neonatal piglets for genotypic validation and identification of genetically engineered “founders” for subsequent breeding and expansion of the novel swine line.